Gut microbiome as a biomarker and therapeutic target for treating obesity or an obesity related disorder

ABSTRACT

The present invention relates to the gut microbiome as a biomarker and therapeutic target for energy harvesting, weight loss or gain, and/or obesity in a subject. In particular, the invention provides methods of altering and monitoring the relative abundance of  Bacteroides  and Firmicutes in the gut microbiome of a subject.

FIELD OF THE INVENTION

The present invention relates to the gut microbiome as a biomarker andtherapeutic target for energy harvesting, weight loss or gain, and/orobesity in a subject.

BACKGROUND OF THE INVENTION

According to the Center for Disease Control (CDC), over sixty percent ofthe United States population is overweight, and greater than thirtypercent are obese. This translates into more than 50 million adults inthe United States with a Body Mass Index (BMI) of 30 or above. Obesityis also a worldwide health problem with an estimated 500 millionoverweight adult humans [body mass index (BMI) of 25.0-29.9 kg/m²] and250 million obese adults (Bouchard, C (2000) N Engl J. Med. 343,1888-9). This epidemic of obesity is leading to worldwide increases inthe prevalence of obesity-related disorders, such as diabetes,hypertension, as well as cardiac pathology, and non-alcoholic fattyliver disease (NAFLD; Wanless, and Lentz (1990) Hepatology 12,1106-1110. Silverman, et al, (1990). Am. J. Gastroenterol. 85,1349-1355; Neuschwander-Tetri and, Caldwell (2003) Hepatology 37,1202-1219). According to the National Institute of Diabetes, Digestiveand Kidney Diseases (NIDDK) approximately 280,000 deaths annually aredirectly related to obesity. The NIDDK further estimated that the directcost of healthcare in the U.S. associated with obesity is $51 billion.In addition, Americans spend $33 billion per year on weight lossproducts. In spite of this economic cost and consumer commitment, theprevalence of obesity continues to rise at alarming rates. From 1991 to2000, obesity in the U.S. grew by 61%.

Although the physiologic mechanisms that support development of obesityare complex, the medical consensus is that the root cause relates to anexcess intake of calories compared to caloric expenditure. While thetreatment seems quite intuitive, dieting is not an adequate long-termsolution for most people; about 90 to 95 percent of persons who loseweight subsequently regain it. Although surgical intervention has hadsome measured success, the various types of surgeries have relativelyhigh rates of morbidity and mortality.

Pharmacotherapeutic principles are limited. In addition, because ofundesirable side effects, the FDA has had to recall several obesitydrugs from the market. Those that are approved also have side effects.Currently, two FDA-approved anti-obesity drugs are orlistat, a lipaseinhibitor, and sibutramine, a serotonin reuptake inhibitor. Orlistatacts by blocking the absorption of fat into the body. An unpleasant sideeffect with orlistat, however, is the passage of undigested oily fatfrom the body. Sibutramine is an appetite suppressant that acts byaltering brain levels of serotonin. In the process, it also causeselevation of blood pressure and an increase in heart rate. Otherappetite suppressants, such as amphetamine derivatives, are highlyaddictive and have the potential for abuse. Moreover, different subjectsrespond differently and unpredictably to weight-loss medications.

Because surgical and pharmacotherapy treatments are problematic, newnon-cognitive strategies are needed to prevent and treat obesity andobesity-related disorders.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a graph showing the effect of decreasing e-value cut-offson EGT assignments to the KEGG database from pyrosequencer and capillarysequencer datasets. Points indicate the average number of KO assignmentsper kb of microbiome sequence. Mean values±s.e.m. are plotted. The GS20pyrosequencer and the 3730 xl capillary sequencer both resulted in anaverage 0.3 KO (KEGG orthology) assignments per kb of sequence at ane-value cutoff <10⁻⁵. However, the number of EGTs present in thepyrosequencer-derived datasets rapidly decays as the e-value cutoff isdecreased, whereas the number of EGTs present in the capillary sequencerdatasets is relatively stable to <10⁻³⁰.

FIG. 2 depicts a graph and tables showing the comparison of datasetsobtained from the cecal microbiomes of obese and lean littermates. (A)Number of observed orthologous groups in each cecal microbiome. Blackindicates the number of observed groups. Grey indicates the number ofpredicted missed groups. (B) Relative abundance of a subset of COGcategories (BLASTX, e-value <10⁻⁵) in the lean1 (pink) and ob1 (blue)cecal microbiome, characterized by capillary- and pyro-sequencers(square, and triangles, respectively). A subset of COG categories (C)and all KEGG pathways (D) consistently enriched or depleted in the cecalmicrobiomes of both obese mice compared to their lean littermates. Reddenotes enrichment and green indicates depletion based on a cumulativebinomial test (brightness indicates level of significance). Blackindicates pathways whose representation is not significantly different.Asterisks indicate groups that were consistently enriched or depletedbetween both sibling pairs using a more stringent EGT assignmentstrategy (e-value <10⁻⁸).

FIG. 3 depicts graphs showing the taxonomic assignments of EGTs and 16SrRNA gene fragments. (A) Relative abundance of EGTs (reads assigned toNR, BLASTX with an e-value <10⁵) in each cecal microbiome confirms thepresence of the indicated bacterial divisions in addition toEuryarcheota. Metazoan sequences (including Mus musculus and fungi) arealso present at low abundance. Bacterial divisions with greater than 1%representation in at least three microbiomes are shown. (B) Alignment of16S rRNA gene fragments (black) confirms our previous PCR-derived 16SrRNA gene sequence-based survey (white). Comparisons include allmicrobiomes sampled with the capillary sequencer (square) and the twomicrobiomes sampled with the pyrosequencer (triangle).

FIG. 4 depicts a graph showing that microbiomes cluster according tohost genotype. (A) Clustering of cecal microbiomes of obese and leansibling pairs based on reciprocal TBLASTX comparisons. All possiblereciprocal TBLASTX comparisons of microbiomes (defined by capillarysequencing) were performed from both lean and obese sibling pairs. Adistance matrix was then created using the cumulative bitscore for eachcomparison and the cumulative score for each self-self comparison.Microbiomes were subsequently clustered using NEIGHBOR(PHYLIP version3.64). (B) Principal Component Analysis (PCA) of KEGG pathwayassignments. A matrix was constructed containing the number of EGTsassigned to each KEGG pathway in each microbiome (includes KEGG pathwayswith >0.6% relative abundance in at least two microbiomes, and astandard deviation >0.3 across all microbiomes), PCA was performed usingCluster3.0, and the results graphed along the first two components.

FIG. 5 depicts KEGG pathways that are enriched or depleted in the cecalmicrobiomes of both obese versus lean sibling pairs, as indicated bybootstrap analysis of relative gene content. Pathways that areconsistently enriched or depleted in the pyrosequencer-based comparisonof ob1 versus leant littermates, and the capillary sequencer-basedcomparison of ob2 versus leant littermates are shown. Red indicatesenrichment and green indicates depletion (brightness denotes level ofsignificance). Black indicates groups that are not significantlychanged.

FIG. 6 depicts graphs showing the biochemical analysis and microbiotatransplantation experiments confirm that the ob/ob microbiome has anincreased capacity for dietary energy harvest (A) Gaschromatography-mass spectrometry quantification of SCFAs in the ceca oflean (black; +/+, ob/+; n=4) and obese (white; ob/ob; n=5)conventionally-raised C57BL/6J mice. (B) Bomb calorimetry of the fecalgross energy content (kcal/g) of lean (black; +/+, ob/+; n=9) and obese(white; ob/ob; n=13) conventionally-raised C57BL/6J mice. (C)Colonization of germ-free wild-type C57BL/6J mice with a cecalmicrobiota harvested from obese donors (white; ob/ob; n=9 recipients)results in a significantly greater percentage increase in total body fatthan colonization with a microbiota from lean donors (black; +/+; n=10recipients). Total body fat content was measured before and after atwo-week colonization, using dual-energy x-ray absorptiometry. Meanvalues±s.e.m. are plotted. Asterisks indicate significant differences(two-tailed Student's t-Test of all datapoints, *p<0.05, **p<0.01,***p<0.001).

FIG. 7 depicts analyses of microbial communities harvested from obese(ob/ob) and lean (+/+) C57BL/6J donor mice and colonized gnotobioticrecipients. Online Unifrac clustering of microbial community structure,based on 4,157 16S rRNA gene sequences (see Table 7 for number ofsequences per sample; ARB tree available atgordonlab.wustl.edu/supplemental/Turnbaugh/obob/). Nodes denoted by ablack square are robust to sequence number (jackknife values >0.70,representing the number of times the node was present when 166 sequenceswere randomly chosen for each mouse for n=100 replicates). Pie chartsindicate the average relative abundance of Firmicutes (black),Bacteroidetes (white), and other (grey; includes Verrucomicrobia,Proteobacteria, Actinobacteria, TM7, and Cyanobacteria) in the donor andrecipient microbial communities.

FIG. 8 depicts a graph of the relative abundance of COG categories(percentage of total EGTs assigned to COG using BLASTX and e-value<10⁻⁵) in the lean1 (black square), ob1 (white square), lean2 (blacktriangle); and ob2 (white triangle) cecal microbiomes. Microbiomes werecharacterized by capillary sequencing.

FIG. 9 depicts COGs that are enriched or depleted in the cecalmicrobiomes of both obese versus can sibling pairs, as indicated bybinomial comparisons of relative gene content. The COGs shown areenriched or depleted in the pyrosequencer-based comparison of ob1 versuslean1 littermates and the capillary sequencer-based comparison of ob2versus lean2 littermates. Red indicates enrichment and green indicatesdepletion (brightness denotes level of significance). Black indicatesgroups that are not significantly changed.

FIG. 10 depicts the correlation between weight loss and gut microbialecology. (A) Clustering of 16S rRNA gene sequence libraries of fecalmicrobiota for each subject (color) and time point (T0=baseline, T1=12weeks, T2=26 weeks, T3=52 weeks of diet therapy) in the two treatmentgroups, based on UniFrac analysis of the 18,348-sequence phylogenetictree. (B) Relative abundance of the Bacteroidetes and Firmicutes. Foreach time point, the values from all available samples were averaged(n=11 or 12 per time point). Lean controls include 4 stool samples fromtwo subjects taken 1 year apart, plus 3 stool samples published. Meanvalues±SE are plotted. (C) Change in Bacteroidetes relative abundanceand weight loss above a threshold of 6% for the CARB-R diet and 2% forthe FAT-R diet.

FIG. 11 depicts an illustration of the experimental design. (A)Diet-induced obesity (DIO) in germ-free mice colonized with a complexmicrobial community. (B) Conventionally-raised (CONV-R) wild-type micefed a Western or CHO diet. (C) Specific dietary shifts after two monthson the Western diet. (D) Microbiota transplantation experiments fromdonor mice on multiple diets to lean germ-free CHO-fed recipients.Numbers in parentheses refer to the age of mice at each step in theprotocol. Mouse diets are labeled Western, FAT-R, CARB-R, and CHO (seeTables 11 and 12).

FIG. 12 depicts data showing that diet-induced obesity alters gutmicrobial ecology in conventionalized mice. Adult C57BL/6Jconventionalized mice were fed a low-fat high-polysaccharide (CHO) orhigh-fat/high-sugar (Western) diet. 16S rRNA gene sequence-based surveyswere performed on the distal gut (cecal) contents of ten mice (n=5mice/group) and the cecal contents from the donor mouse. UniFrac-basedanalysis of community membership (who's there) indicates that thecommunities cluster based on diet: the community from CHO fed recipientsclusters with the CHO fed donor cecal microbiota, whereas the communityfrom Western diet fed recipients has been altered. Black boxes indicatenodes that were reproduced in >70% of all jackknife replications (n=96sequences). The relative abundance of the Firmicutes is increased in theWestern diet microbiota, corresponding to a bloom in the Mollicutesclass. Pie charts show the average relative abundance of bacteriallineages in the CHO diet versus Western diet cecal microbiota (n=5mice/group). The asterisk indicates that the sample was also analyzedbased on whole community shotgun sequencing.

FIG. 13 depicts graphs showing that diet-induced obesity (DIO) is linkedto changes in gut microbial ecology, resulting in an increased capacityof the distal gut microbiota to promote host adiposity. (A) The relativeabundance (% of total 16S rRNA gene sequences) of the Firmicutes andBacteroidetes divisions in the distal gut (cecal) microbiota ofconventionalized, wild-type C57BL/6J mice fed a standard low-fathigh-polysaccharide chow diet (CHO; n=5) or a high-fat/high-sugarWestern diet (n=5). (B) DIO is associated with a marked reduction in theoverall diversity of the cecal bacterial community. The Shannon index ofdiversity was calculated at multiple phylotype cutoffs (defined by %identity of 16S rRNA gene sequences) for each individual cecal datasetusing DOTUR [13]. The average diversity at each cutoff is plotted formice fed the CHO and Western diets. (C) DIO is linked to a bloom of theMollicutes class of bacteria within the Firmicutes division. Therelative abundance of the Mollicutes is shown for conventionalized micefed the CHO or Western diet. (D) Microbiota transplantation experimentsreveal that the DIO community has an increased capacity to promote hostfat deposition. Total body fat was measured using dual-energy x-rayabsorptiometry (DEXA) before and after a two-week colonization of adultgerm-free CHO-fed C57BL/6J wild-type mice with a cecal microbiotaharvested from mice maintained on CHO or Western diet (n=14mice/treatment group). Mean values±SEM are shown. Asterisks in panelsA-D indicate that the differences are statistically significant(Student's t-test, p<0.05), after using the Bonferroni correction tolimit false positives.

FIG. 14 depicts the phylogeny of selected representatives from theFirmicutes division, including the Mollicute bloom and closely relatedhuman strains. 16S rRNA gene sequences for previously sequencedFirmicute genomes and Mollicute strains isolated from the human gut wereidentified in the RDP database [34]. All Mollicute sequences obtainedfrom conventionalized C57BL/6J mice fed a CHO or Western diet (n=801sequences) and from our previous survey of obese humans (length >1250nucleotides; n=571 sequences) [9] were separately binned into phylotypesusing DOTUR (99% identity) [13]. One representative of each of the sixdominant mouse phylotypes was chosen (together comprising 81% of themouse Mollicute sequences) in addition to one representative of each ofthe ten dominant human phylotypes. Likelihood parameters were determinedusing Modeltest [35] and a maximum-likelihood tree was generated usingPAUP [36]. Bootstrap values represent nodes found in >70 of 100repetitions. Phylotypes from the Mollicute bloom are shown in blue;wedge size is proportional to the indicated relative abundance (% ofMollicute 16S rRNA gene sequences). The Mollicute bloom and relativesare shaded in blue, previously sequenced Mollicutes (including theobligate parasites, Mycoplasma, and Mesoplasma florum) are shaded inyellow, and recently sequenced Firmicutes found in the normal distalhuman gut microbiota are shaded in red. Akkermansia muciniphila, aVerrucomicrobia, was used to root the tree (shaded in green).

FIG. 15 depicts a graph showing the Mollicute bloom occurs inconventionally-raised wild-type C57BL/6J mice as well as in mice withoutan intact innate or adaptive immune system. Wild-type (+/+), MyD88−/−,or Rag1−/− C57BL/6J mice were weaned onto a standard low-fatpolysaccharide-rich (CHO) or high-fat/high-sugar (Western) diet. 16SrRNA gene sequence-based surveys were performed; sequences were aligned[41], and inserted into an ARB neighbor-joining tree [42]. Asterisksindicate significant differences (Student's t-test p<0.001).

FIG. 16 depicts a graph showing mice with diet-induced obesity that areswitched to a FAT-R or CARBR diet exhibit stabilization of weight,decreased caloric intake and reduced adiposity. (A) Weight gain (g) and(B) percentage epidydymal fat-pad weight to body weight in wild typeC57BL/6J mice that were initially weaned onto a Western diet for 8weeks, and then maintained on the Western diet, or switched to a FAT-Ror CARB-R diet for four weeks (n=5-6 mice/treatment group). Weight wasmonitored during the four week period. (C) Chow consumption (kcal/d) isdecreased in mice switched to a FAT-R or CARB-R diet. Data arerepresented as mean±SEM. Asterisks indicate significant differences(ANOVA of FAT-R or CARB-R versus Western, *p<0.05, **p<0.01,***p<0.0001).

FIG. 17 depicts data showing that switching from a Western to FAT-R orCARB-R diet results in a division-wide increase in the relativeabundance of Bacteroidetes, and a decrease in the relative abundance ofMollicutes. UniFrac-based analysis of bacterial community membershipshows an impact of diet on gut microbial ecology: cecal communitiesanalyzed from two families of C57BL/6J wild-type mice (Table 13)generally cluster based on host diet (Western, FAT-R, and CARB-R). Theaverage relative abundance (% of total 16S rRNA gene sequences) ofbacterial lineages within the cecal microbiota of all mice fed aWestern, FAT-R, or CARB-R diet is displayed as pie charts. Black boxesindicate nodes that were reproduced in >50% of all jackknifereplications (n=126 sequences were randomly re-sampled). Asterisksindicate cecal samples that were analyzed by whole community shotgunsequencing.

FIG. 18 depicts charts showing the taxonomic assignments of metagenomicsequencing reads from seven cecal microbiome datasets based on BLASThomology searches, and by alignment of 16S rRNA gene fragments. (A) Thececal microbiome is dominated by sequences homologous to Bacteria.Sequencing reads were trimmed based on quality and vector sequence andthe resulting datasets were used as queries against the NCBInon-redundant database (e-value <10⁻⁵). Sequences were assigned to thelowest taxonomic group that would include all significant hits, usingMEGAN [18]. Pie charts are shown for each individual dataset and for theaverage of all datasets. Colors indicate assignments to bacteria (red),archaea (green), eukarya (yellow), viruses (blue), sequences that couldnot be confidently assigned to a group (purple), and sequences with nosignificant BLASTX matches (orange). (B) Relative abundance ofmicrobiome sequences homologous to genomes from four bacterialdivisions: Bacteroidetes (red), Proteobacteria (yellow), Actinobacteria(orange), and Firmicutes (blue). All divisions observed at >1% relativeabundance are shown. (C) Relative abundance of microbiome sequenceshomologous to genomes from bacterial classes within the Firmicutesdivision: Bacilli (dark blue), Clostridia (yellow), and Mollicutes(light blue). (D) Taxonomic assignments of 16S rRNA gene fragmentsobtained from cecal microbiome datasets. 16S rRNA gene fragments wereidentified by querying the Ribosomal Database Project (RDP) database(version 9.33; BLASTN e-value <10⁻⁵) [34]. 16S rRNA gene fragments werealigned with NAST [41] and added to an ARB neighbor-joining tree [42].16S rRNA gene fragments from the Bacteroidetes (red), Proteobacteria(yellow), Verrucomicrobia (green), Mollicutes (light blue), and otherFirmicutes (dark blue) are shown.

FIG. 19 depicts an illustration showing the metabolic reconstructions ofthe Eubacterium dolichum genome and the Western diet microbiome.Predicted gene presence calls for the Western diet microbiome and/or theE. dolichum genome are displayed in the upper right. Fermentationend-products and cellular biomass are highlighted in white ellipses.Note that culture based studies of E. dolichum have demonstrated itsability to produce lactate, acetate, and butyrate [37], suggesting thatthe apparent gap in the pathway for generating butyrate reflects thedraft nature of the genome assembly or the possibility that thisorganism uses novel enzymes to generate this end-product of anaerobicfermentation. Abbreviations for enzymes (in boldface): Pg1,phosphoglucose isomerase; Pfk, phosphofructokinase; Fba,fructose-1,6-bisphosphate aldolase; Tpi, triose-phosphate isomerase;Gap, glyceraldehyde-3-phosphate dehydrogenase; Pgk, phosphoglyceratekinase; Pgm, phosphoglycerate mutase; Eno, enolase; Pyk, pyruvatekinase; EI, PTS enzyme I; HPr, PTS protein HPr; EIIA/B/C, PTS proteins;DXPS, 1-deoxy-D-xylulose-5-phosphate synthase; DXPR,DXP-reductoisomerase; MEPC, MEP cytidylyltransferase; MEK, CDPME kinase;MECS, MECDP-synthase; MDPS, 4-hydroxy-3-methylbut-2-en-1-yl diphosphatesynthase; MDPR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; Ldh,L-lactate dehydrogenase; Pfl, pyruvate formate-lyase; Pat, phosphateacetyltransferase; Ak, acetate kinase; Aca, acetyl-CoAC-acetyltransferase; Bhbd, 3-hydroxybutyryl-CoA dehydrogenase; Ech,enoyl-CoA hydratase; Bcd, butyryl-CoA dehydrogenase; Ptb,phosphotransbutyrylase; Bk, butyrate kinase; 1-Pfk,1-phosphofructokinase; Npd, N-acetylglucosamine-6-phosphate deacetylase;Gpi, phosphoglucosamine isomerase; Fbf, fructan beta-fructosidase.

FIG. 20 depicts an illustration showing the assembly of metagenomicsequence data reveals physical linkage between the Mollicutephosphotransferase system (PTS) and other genes involved in carbohydratemetabolism. The pooled mouse gut microbiome dataset was assembled usingARACHNE [24] (n=7 combined datasets; see Tables S6 and S7 for assemblystatistics). The contig length is shown as a solid black bar. Arrowsindicate predicted proteins. Functional assignments were derived fromthe NCBI annotations and verified by BLASTP comparisons of eachpredicted protein with the STRING-extended COG database [19] and theKEGG database [20], in addition to Hidden Markov Model (HMM)-basedprotein domain searching with InterProScan [31]. Contigs 23 and 73are >98% identical over the region in pink (234/238 nucleotides): theyare likely different ends of the same gene that were not joined due tothe relatively stringent assembly parameters employed.

FIG. 21 depicts a graph showing the concentration of bacterialfermentation end-products in the ceca of Western, FAT-R, and CARB-Rmice. Acetate and butyrate levels (μmol per g wet weight cecal contents)were measured by gas chromatography mass spectrometry. Lactate levels(mM per kg protein) were measured using established microanalyticmethods (see Examples). Data are represented as mean±SEM. Asterisksindicate significant differences (Student's t-test of Western versusCARB-R, *p<0.05, **p<0.01).

FIG. 22 depicts graphs showing principal component analysis (PCA) ofsequenced Firmicute genomes. (A) PCA analysis of 14 previously sequencedMollicute genomes (mostly Mycoplasma) and draft genome assemblies ofnine human gut-associated Firmicutes (genome.wustl.edu/pub/). MetaGenewas used to predict proteins from each genome [25]. Proteins were thenassigned to KEGG orthologous groups based on homology (BLASTP e-value<10⁻⁵; KEGG version 40) [20]. Genomes were clustered based on therelative abundance of KEGG metabolic pathways (number of assignments toa given pathway divided by total number of pathway assignments). Onlypathways found at >0.6% relative abundance in at least two genomes wereincluded. The first two components are shown, representing 17% and 8% ofthe variance respectively. Abbreviations: Mca, Mycoplasma capricolum;Mfl, Mesoplasma florum L1; Mga, Mycoplasma gallisepticum R, Mge,Mycoplasma genitalium G37; Mhy232, Mycoplasma hyopneumoniae 232;Mhy7448, Mycoplasma hyopneumoniae 7448; MhyJ, Mycoplasma hyopneumoniaeJ; Mmo, Mycoplasma mobile 163K; Mmy, Mycoplasma mycoides subsp. mycoidesSC str. PG1; Mpe, Mycoplasma penetrans HF-2; Mpn, Mycoplasma pneumoniaeM129; Mpu, Mycoplasma pulmonis UAB CTIP; Msy, Mycoplasma synoviae 53;Upa, Ureaplasma parvum; E. dolichum, Eubacterium dolichum; CL250,Clostridium sp. L2-50; C. symbiosum, Clostridium symbiosum; Dlo, Dorealongicatena; Eel, Eubacterium eligens; Ere, Eubacterium rectale; Eve,Eubacterium ventriosum; Rob, Ruminococcus obeum; and Rto, Ruminococcustorques. (B) KEGG pathway relative abundance has a significantcorrelation with genome size. A linear regression was performedcomparing PCA1 to genome size (or draft assembly size). PCA1 has asignificant correlation to genome size (R2=0.9, p<0.05). (C) Metabolicpathways in E. dolichum. Pathways are marked partial if most genes arepresent and absent if s2 genes are present.

FIG. 23 depicts the KEGG metabolic pathways significantly enriched inthe human gut-derived Eubacterium dolichum strain DSM 3991 genomerelative to eight human gut-associated Firmicutes. Pathways whoserelative representation is significantly different between the E.dolichum genome and the pooled gut Firmicute genomes (n=8) wereidentified using a bootstrap comparison of the abundance of sequencesassigned to all KEGG pathways (xipe version 2.4; confidence level=0.98,sample size=10,000) [32]. The relative abundance of all KEGG pathwayswith significantly different representation found at a relativeabundance >0.6% in at least two microbiome datasets was transformed intoa z-score and clustered by genome and pathway using a Euclidean distancemetric [47]. Enrichment (yellow) and depletion (blue) are defined as arelative abundance greater or less than the mean for all datasets (i.e.a z-score greater or less than zero, respectively). For full strainnames see FIG. 22.

FIG. 24 depicts a STRING-based protein network analysis of the predictedE. dolichum proteome. MetaGene [25] was used to predict proteins fromthe E. dolichum deep draft assembly. Proteins were subsequently assignedto COGs based on homology (BLASTP e-value <10⁻⁵) [19]. Annotated COGinteractions were used to organize the protein network, includinginteractions based on neighborhood, gene fusion, co-occurrence,homology, co-expression, experiments, databases, and text mining (MedusaJava appet) [38]. Nodes, each representing a different orthologousgroup, are colored as follows: green, present in all analyzed Firmicutegenomes (including the mycoplasma); blue, present in all recentlysequenced gut Firmicute genomes; red, present in the Westerndietassociated cecal microbiome (based on BLAST homology searches,e-value <10⁻⁵ and the deposited annotations in the STRING database,version 7). 89% of the COGs found in the E. dolichum genome were alsofound in the Western diet microbiome. Most of the COGs in green areinvolved in essential cellular functions such as transcription andtranslation (56% of the COG category assignments are to ‘Informationstorage and processing’). Some clusters of interest are highlighted,including the phosphotransferase system (PTS), the 2-methyl-D-erythritol4-phosphate pathway for isoprenoid biosynthesis (MEP), cell wallbiosynthesis, ABC transporters, and V-type ATPases for H⁺ import.

SUMMARY OF THE INVENTION

One aspect of the present invention encompasses a method for decreasingenergy harvesting, decreasing body fat, or for promoting weight loss ina subject. The method comprises altering the microbiota population inthe subject's gastrointestinal tract by increasing the relativeabundance of Bacteroidetes.

Another aspect of the invention encompasses a composition comprising anantibiotic having efficacy against Firmicutes but not againstBacteroidetes, and a probiotic comprising Bacteroidetes.

Yet another aspect of the invention encompasses a method for selecting acompound for treating obesity or an obesity-related disorder in a host.The method comprises providing a microbiome profile from the host andproviding a plurality of reference microbiome profiles, each associatedwith a compound. The host profile and each reference profile has aplurality of values, each value representing the abundance of amicrobiome biomolecule. The method further comprises selecting thereference profile most similar to the host microbiome profile, therebyselecting a compound for treating obesity or an obesity-related disorderin the host.

Still another aspect of the invention encompasses a method to determinewhether a compound has efficacy for treatment of obesity or anobesity-related disorder in a host. The method comprises comparing aplurality of biomolecules of the host's microbiome before and afteradministration of a drug for the treatment of obesity, such that if theabundance of biomolecules associated with obesity decreased aftertreatment, the compound is efficacious in treating obesity in a host.

An additional aspect of the invention encompasses a method of predictingrisk for obesity or an obesity-related disorder in a host. The methodcomprises providing a microbiome profile from said host and providing aplurality of reference microbiome profiles. The host profile and eachreference profile has a plurality of values, each value representing theabundance of a microbiome biomolecule. The method further comprisesselecting the reference profile most similar to the host microbiomeprofile, such that if the host's microbiome is most similar to areference obese microbiome, the host is at risk for obesity or anobesity-related disorder.

Another additional aspect of the invention encompasses acomputer-readable medium comprising a plurality of digitally encodedprofiles wherein each profile of the plurality has a plurality ofvalues, each value representing the abundance of a biomolecule in anobese host microbiome.

A further aspect of the invention encompasses a kit for evaluating adrug, or for diagnosing or prognosing a gut microbiome associated withincreased energy harvesting, increased body fat, and/or weight gain. Thekit comprises an array comprising a substrate, the substrate havingdisposed thereon at least one biomolecule that is modulated in an obesehost microbiome compared to a lean host microbiome, and acomputer-readable medium having a plurality of digitally-encodedprofiles wherein each profile of the plurality has a plurality ofvalues, each value representing the abundance of biomolecule in a hostmicrobiome detected by the array.

Another further aspect of the invention encompasses at method fordecreasing body fat or for promoting weight loss in a subject. Themethod comprising altering the activity of the microbiota population inthe subject's gastrointestinal tract by altering the microbiotapopulation.

Other aspects and iterations of the invention are described morethoroughly below.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered, as demonstrated in the Examples, that there is arelationship between the diversity of the gut microbiota and obesity. Inparticular, an obese subject typically has fewer Bacteroidetes and moreFirmicutes compared to a lean subject. Taking advantage of thesediscoveries, the present invention provides compositions and methods toregulate energy balance in a subject. The invention also provides toolsutilizing the gut microbiome as a diagnostic or prognostic biomarker forobesity risk, a biomarker for drug discovery, a biomarker for thediscovery of therapeutic targets involved in the regulation of energybalance, and a biomarker for the efficacy of a weight loss program.

I. Modulation of Energy Balance in a Subject

The energy balance of a subject may be modulated by altering thesubject's gut microbiota population. Generally speaking, to decreaseenergy harvesting, decrease body fat, or promote weight loss, therelative abundance of bacteria within the Bacteroidetes division isincreased and optionally, the relative abundance of bacteria within theFirmicutes division is decreased. Alternatively, to increase energyharvesting, to increase body fat, or promote weight gain, the relativeabundance of Bacteroidetes is decreased and optionally, the relativeabundance of Firmicutes is increased. Additional agents may also beutilized to achieve either weight loss or weight gain. Examples of theseagents are detailed in section I(c).

(a) Altering the Abundance of Bacteroides and/or Firmicutes

The relative abundance of Bacteroidetes may be altered by increasing ordecreasing the presence of one or more Bacteroidetes species that residein the gut. Non-limiting examples of species may include the specieslisted in Table A. Additionally, non-limiting examples of species mayinclude B. thetaiotaomicron, B. vulgatus, B. ovatus, B. distasonis, B.uniformis, B. stercoris, B. eggerthii, B. merdae, and B. caccae. In oneembodiment, the population of B. thetaiotaomicron is altered. In stillanother embodiment, the population of B. vulgatus is altered. In anadditional embodiment, the population of B. ovatus is altered. Inanother embodiment, the population of B. distasonis is altered. In yetanother embodiment, the population of B. uniformis is altered. In anadditional embodiment, the population of B. stercoris is altered. In afurther embodiment, the population of B. eggerthii is altered. In stillanother embodiment, the population of B. merdae is altered. In anotherembodiment, the population of B. caccae is altered. In a furtherembodiment, the species within the division Bacteroidetes may be as ofyet unnamed.

TABLE A Number Divisions Genus Species Strain ID 1 BacteroidetesAlistepes putredinis ATCC 29800 2 Bacteroidetes Bacteroides caccae ATCC43185T 3 Firmicutes Clostridium leptum ATCC 29065 4 FirmicutesClostridium boltaea ATCC BAA-613 5 Firmicutes Peptostreptococcus microsATCC 33270 6 Firmicutes Eubacterium ventriosum ATCC 27560 7 FirmicutesEubacterium halii ATCC 27751 8 Firmicutes Ruminococcus gnavus ATCC 291499 Firmicutes Coprococcus catus ATCC 27761 10 Firmicutes Eubacteriumsiraeum ATCC 29066 11 Firmicutes Ruminococcus obeum ATCC 29174 12Firmicutes Ruminococcus torques ATCC 27756 13 Firmicutes Subdoligranulumvariabile CCUG 47106 14 Firmicutes Dorea formicigenerans ATCC 27755 15Firmicutes Dorea longicatena CCUG 45247 16 Firmicutes Faecalibacteriumprausnitzii ATCC 27768 17 Bacteroidetes Bacteroides sp. CCUG 39913 18Bacteroidetes Bacteroides sp. Smarlab 3301186 19 BacteroidetesBacteroides ovatus ATCC 8483T 20 Bacteroidetes Bacteroides salyersiaeATCC BAA-997 21 Bacteroidetes Alistepes finegoldii CCUG 46020 22Bacteroidetes Bacteroides sp. MPN isolate group 6 23 BacteroidetesBacteroides sp. DSM 12148 24 Bacteroidetes Bacteroides merdae ATCC43184T 25 Bacteroidetes Bacteroides stercosis ATCC 43183T 26Bacteroidetes Bacteroides uniformis ATCC 8492 27 BacteroidetesBacteroides WH302 Gordon Lab 28 Firmicutes Bulleidia moorei ATCC BAA-17029 Firmicutes Bacteroides capillosus ATCC 29799 30 FirmicutesRuminococcus bromii ATCC 27255 31 Firmicutes Clostridium symbiosum ATCC14940 32 Firmicutes Clostridium sp. DSM 6877(FS41) 33 FirmicutesClostridium sp. A2-207 34 Firmicutes Anaerofustis stercorihominis CCUG47767T 35 Firmicutes Clostridium scindens ATCC 35704 36 FirmicutesClostridium spiroforme DSM 1552 37 Firmicutes Ruminococcus callidus ATCC27760 38 Firmicutes Coprococcus eutactus ATCC 27759 39 FirmicutesGemella haemolysans ATCC 10379 40 Firmicutes Clostridium sp. A2-183 41Firmicutes Clostridium sp. SL6/1/1 42 Firmicutes Roseburia intestinalisDSM 14610 43 Firmicutes Clostridium sp. GM2/1 44 Firmicutes Clostridiumsp. A2-194 45 Firmicutes Clostridium sp. 14774 46 Firmicutes Clostridiumsp. A2-166 47 Firmicutes Clostridium sp. A2-175 48 Firmicutes Roseburiafaecalis M6/1 49 Firmicutes Catenibacterium mitsuokai JCM 10609 50Firmicutes Clostridium sp. SR1/1 51 Firmicutes Clostridium sp. L1-83 52Firmicutes Clostridium sp. L2-6 53 Firmicutes Clostridium sp. A2-231 54Firmicutes Clostridium sp. A2-165 55 Firmicutes Dialister sp. E2_20 56Firmicutes Clostridium sp. SS2/1 57 Firmicutes Anaerotruncus colihominisCCUG 45055T 58 Firmicutes Eubacterium plautii ATCC 29863 59 FirmicutesClostridium bartlettii CCUG 48940 60 Firmicutes Lactobacilllus lactisSsp. IL1403

The present invention also includes altering various combinations ofspecies, such as at least two species, at least three species, at leastfour species, at least five species, at least six species, at leastseven species, at least eight species, at least nine species, or atleast ten species. For example, the combination of B. thetaiotaomicron,B. vulgatus, B. ovatus, B. distasonis, and B. uniformis may be altered.

In an exemplary embodiment, the relative abundance of Bacteroidetes isincreased to decrease energy harvesting, decrease body fat, or promoteweight loss in a subject. Increased abundance of Bacteroidetes in thegut may be accomplished by several suitable means generally known in theart. In one embodiment, a food supplement that increases the abundanceof Bacteroidetes may be administered to the subject. By way of example,one such food supplement is psyllium husks as described in U.S. PatentApplication Publication No. 2006/0229905, which is hereby incorporatedby reference in its entirety. In an exemplary embodiment, a probioticcomprising Bacteroidetes may be administered to the subject. The amountof probiotic administered to the subject can and will vary dependingupon the embodiment. The probiotic may be present at a level of fromabout one thousand to about ten billion cfu/g (colony forming units pergram) of the total composition or of the part of the compositioncomprising the probiotic. In one embodiment, the probiotic may bepresent at a level of from about one hundred million to about 10 billionorganisms. The probiotic microorganism may be in any suitable form, forexample in a powdered dry form. In addition, the probiotic microorganismmay have undergone processing in order for it to increase its survival.For example, the microorganism may be coated or encapsulated in apolysaccharide, fat, starch, protein or in a sugar matrix. Standardencapsulation techniques known in the art can be used, and for example,as discussed in U.S. Pat. No. 6,190,591, which is hereby incorporated byreference in its entirety.

Alternatively, the relative abundance of Bacteroidetes is decreased toincrease energy harvesting, increase body fat, or promote weight gain ina subject. Decreased abundance of Bacteroidetes in the gut may beaccomplished by several suitable means generally known in the art. Inone embodiment, an antibiotic having efficacy against Bacteroidetes maybe administered. Generally speaking, antimicrobial agents may targetseveral areas of bacterial physiology: protein translation, nucleic acidsynthesis, folic acid metabolism, or cell wall synthesis. In anexemplary embodiment, the antibiotic will have efficacy againstBacteriodetes but not against Firmicutes. The susceptibility of thetargeted species to the selected antibiotics may be determined based onculture methods or genome screening.

It is contemplated that the abundance of gut Bacteroidetes within anindividual subject may be altered (i.e., increased or decreased) fromabout a one fold difference to about a ten fold difference or more,depending on the desired result (i.e., increased energy harvesting(weight gain) or decreased energy harvesting (weight loss)) and theindividual subject. In one embodiment, the abundance may be altered fromabout a one fold difference to about a ten fold difference. For weightloss, the abundance may be altered by an increase of about a two folddifference to about a ten fold difference, of about a three folddifference to about a ten fold difference, of about a four folddifference to about a ten fold difference, of about a five folddifference to about a ten fold difference, or of about a six folddifference to about a ten fold difference. A method for determining therelative abundance of gut Bacteroidetes is described in the examples,alternatively, an array of the invention, described below, may be usedto determine the relative abundance.

Stated another way, it is contemplated that the abundance of gutBacteroidetes within an individual subject may be altered (i.e.,increased or decreased) from about 1% to about 100% or more depending onthe desired result (i.e., increased energy harvesting (weight gain) ordecreased energy harvesting (weight loss)) and the individual subject.For weight loss, the abundance may be altered by an increase of fromabout 20% to about 100%, from about 30% to about 100%, from about 40% toabout 100%, from about 50% to about 100%, from about 60% to about 100%,from about 70% to about 100%, from about 80% to about 100%, or fromabout 90% to 100%. A method for determining the relative abundance ofgut Bacteroidetes is described in the examples, alternatively, an arrayof the invention, described below, may be used to determine the relativeabundance.

(b) Altering the abundance of Firmicutes

The relative abundance of Firmicutes may be altered by increasing ordecreasing the presence of one or more species that reside in the gut.Non-limiting examples of species may include the species listed in TableA Representative species include species from Clostridia, Bacilli, andMollicutes. In one embodiment, the relative abundance of one or moreClostridia species is altered. In another embodiment, the relativeabundance of one or more Bacilli species is altered. In yet anotherembodiment, the relative abundance of one or more Mollicutes species isaltered. It is also contemplated that the relative abundance of severalspecies of Firmicutes may be altered without departing from the scope ofthe invention. By way of non-limiting examples, a combination of one ormore Clostridia species, one or more Bacilli species, and one or moreMollicutes species may be altered. In a further embodiment, the specieswithin the division Firmicutes may be as of yet unnamed.

In some embodiments, the Mollicutes class is altered. For instance, E.dolichum, E. cylindroides, or E. biforme may be altered. In oneembodiment, the species of the Mollicutes class may posses the geneticinformation to create a cell wall. In another embodiment, the species ofthe Mollicutes class may produce a cell wall. In a further embodiment,the species within the class Mollicutes may be as of yet unnamed.

In an exemplary embodiment, the relative abundance of Firmicutes isdecreased to decrease energy harvesting, decrease body fat, or promoteweight loss in a subject. Decreased abundance of Firmicutes in the gutmay be accomplished by several suitable means generally known in theart. In one embodiment, an antibiotic having efficacy against Firmicutesmay be administered. In an exemplary embodiment, the antibiotic willhave efficacy against Firmicutes but not against Bacteriodetes. Inanother exemplary embodiment, the antibiotic will have efficacy againstMollicutes, but not Bacteriodetes. The susceptibility of the targetedspecies to the selected antibiotics may be determined based on culturemethods or genome screening.

Alternatively, the relative abundance of Firmicutes is increased toincrease energy harvesting, increase body fat, or promote weight gain ina subject. Increased abundance of Firmicutes in the gut may beaccomplished by several suitable means generally known in the art. In anexemplary embodiment, a probiotic comprising Firmicutes may beadministered to the subject.

It is contemplated that the abundance of gut Firmicutes may be altered(i.e., increased or decreased) from about a one fold difference to abouta ten fold difference or more, depending on the desired result (i.e.,increased energy harvesting (weight gain) or decreased energy harvesting(weight loss)). For weight loss, the abundance may be altered by adecrease of about a one fold difference to about a ten fold difference,a two fold difference to about a ten fold difference, of about a threefold difference to about a ten fold difference, of about a four folddifference to about a ten fold difference, of about a five folddifference to about a ten fold difference, or of about a six folddifference to about a ten fold difference. A method for determining therelative abundance of gut Firmicutes is described in the examples.

Stated another way, it is contemplated that the abundance of gutFirmicutes may be altered (i.e., increased or decreased) from about 1%to about 100% or more depending on the desired result (i.e., increasedenergy harvesting (weight gain) or decreased energy harvesting (weightloss)). For weight loss, the abundance may be altered by a decrease offrom about 20% to about 100%, from about 30% to about 100%, from about40% to about 100%, from about 50% to about 100%, from about 60% to about100%, from about 70% to about 100%, from about 80% to about 100%, orfrom about 90% to 100%. A method for determining the relative abundanceof gut Firmicutes is described in the examples.

(c) Additional Weight Modulating Agents

Another aspect of the invention encompasses a combination therapy toregulate fat storage, energy harvesting, and/or weight loss or gain in asubject. In an exemplary embodiment, a combination for decreasing energyharvesting, decreasing body fat or for promoting weight loss isprovided. For this embodiment, a composition comprising an antibiotichaving efficacy against Firmicutes but not against Bacteroidetes; and aprobiotic comprising Bacteroidetes may be administered to the subject.Additionally, an anti-archea compound may be included in theaforementioned composition. Other agents that may be included with theaforementioned composition are detailed below.

The compositions utilized in this invention may be administered by anynumber of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, or rectal means. The actualeffective amounts of compounds comprising a weight loss composition ofthe invention can and will vary according to the specific compoundsbeing utilized, the mode of administration, and the age, weight andcondition of the subject. Dosages for a particular individual subjectcan be determined by one of ordinary skill in the art using conventionalconsiderations. Those skilled in the art will appreciate that dosagesmay also be determined with guidance from Goodman & Gilman's ThePharmacological Basis of Therapeutics, Ninth Edition (1996), AppendixII, pp. 1707-1711 and from Goodman & Gilman's The Pharmacological Basisof Therapeutics, Tenth Edition (2001), Appendix II, pp. 475-493.

i. Fiaf Polypeptide

A composition of the invention for promoting weight loss may optionallyinclude either increasing the amount of a Fiaf polypeptide or theactivity of a Fiaf polypeptide. Typically, a suitable Fiaf polypeptideis one that can substantially inhibit LPL when administered to thesubject. Several Fiaf polypeptides known in the art are suitable for usein the present invention. Generally speaking, the Fiaf polypeptide isfrom a mammal. By way of non-limiting example, suitable Fiafpolypeptides and nucleotides are delineated in

TABLE B TABLE B Species PubMed Ref. Homo sapiens NM_139314 NM_016109 Musmusculus NM_020581 Rattus norvegicus NM_199115 Sus scrofa AY307772 Bostaurus AY192008 Pan troglodytes AY411895

In certain aspects, a polypeptide that is a homolog, ortholog, mimic ordegenerative variant of a Fiaf polypeptide is also suitable for use inthe present invention. In particular, the subject polypeptide willtypically inhibit LPL when administered to the subject. A variety ofmethods may be employed to determine whether a particular homolog, mimicor degenerative variant possesses substantially similar biologicalactivity relative to a Fiaf polypeptide. Specific activity or functionmay be determined by convenient in vitro, cell-based, or in vivo assays,such as measurement of LPL activity in white adipose tissue or in theheart. In order to determine whether a particular Fiaf polypeptideinhibits LPL, the procedure detailed in the examples of U.S. PatentApplication No. 20050239706, which is hereby incorporated by referencein its entirety, may be followed.

Fiaf polypeptides suitable for use in the invention are typicallyisolated or pure and are generally administered as a composition inconjunction with a suitable pharmaceutical carrier, as detailed below. Apure polypeptide constitutes at least about 90%, preferably, 95% andeven more preferably, at least about 99% by weight of the totalpolypeptide in a given sample.

The Fiaf polypeptide may be synthesized, produced by recombinanttechnology, or purified from cells using any of the molecular andbiochemical methods known in the art that are available for biochemicalsynthesis, molecular expression and purification of the Fiafpolypeptides [see e.g., Molecular Cloning, A Laboratory Manual(Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols inMolecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc.,Wiley-Interscience, New York)].

The invention also contemplates use of an agent that increases Fiaftranscription or its activity. For example, an agent may be deliveredthat specifically activates Fiaf expression: this agent may be a naturalor synthetic compound that directly activates Fiaf gene transcription,or indirectly activates expression through interactions with componentsof host regulatory networks that control Fiaf transcription. Suitableagents may be identified by methods generally known in the art, such asby screening natural product and/or chemical libraries using thegnotobiotic zebrafish model described in the examples of U.S. PatentApplication No. 20050239706. In another embodiment, a chemical entitymay be used that interacts with Fiaf targets, such as LPL, to reproducethe effects of Fiaf (e.g., in this case inhibition of LPL activity). Inan alternative of this embodiment, administering a Fiaf agonist to thesubject may increase Fiaf expression and/or activity. In one embodiment,the Fiaf agonist is a peroxisome proliferator-activated receptor (PPARs)agonist. Suitable PPARs include PPARα, PPARβ/δ, and PPARγ. Fenofibrateis another suitable example of a Fiaf agonist. Additional suitable Fiafagonists and methods of administration are further described in Manards,et al., J. Biol Chem, 279, 34411 (2004), and U.S. Patent Publication No.2003/0220373, which are both hereby incorporated by reference in theirentirety.

ii. Other Compounds

The compositions of the invention that decrease energy harvesting,decrease body fat, or promote weight loss may also include severaladditional agents suitable for use in weight loss regimes. Generallyspeaking, exemplary combinations of therapeutic agents may actsynergistically to decrease energy harvesting, decrease body fat, orpromote weight loss. Using this approach, one may be able to achievetherapeutic efficacy with lower dosages of each agent, thus reducing thepotential for adverse side effects. In one embodiment, acarbose may beadministered with a composition of the invention. Acarbose is aninhibitor of α-glucosidases and is required to break down carbohydratesinto simple sugars within the gastrointestinal tract of the subject. Inanother embodiment, an appetite suppressant, such as an amphetamine, ora selective serotonin reuptake inhibitor, such as sibutramine, may beadministered with a composition of the invention. In still anotherembodiment, a lipase inhibitor such as orlistat, or an inhibitor oflipid absorption such as Xenical, may be administered with a compositionof the invention.

iii. Restricted Calorie Diet

Optionally, in addition to administration of a composition of theinvention for weight loss, a subject may also be placed on a restrictedcalorie diet. As shown in the example, restricted calorie diets arehelpful for increasing the relative abundance of Bacteroidetes anddecreasing the relative abundance of Firmicutes. Several restrictedcalorie diets known in the art are suitable for use in combination withthe compositions of the invention. Representative diets include areduced fat diet, reduced protein, or a reduced carbohydrate diet.

iv. Alteration of the Gastrointestinal Archaeon Population

An anti-archea compound may be included in a composition of theinvention to decrease energy harvesting, decrease fat storage, and/ordecrease weight gain. To promote weight loss in a subject, the archaeonpopulation is altered such that microbial-mediated carbohydratemetabolism or its efficiency is decreased in the subject, wherebydecreasing microbial-mediated carbohydrate metabolism or its efficiencypromotes weight loss in the subject.

Accordingly, in one embodiment, the subject's gastrointestinal archaeonpopulation is altered so as to promote weight loss in the subject.Typically, the presence of at least one genera of archaeon that residesin the gastrointestinal tract of the subject is decreased. In mostembodiments, the archaeon is generally a mesophilic methanogenicarchaea. In one alternative of this embodiment, the presence of at leastone species from the genera Methanobrevibacter or Methanosphaera isdecreased. In another alternative embodiment, the presence ofMethanobrevibacter smithii is decreased. In still another embodiment,the presence of Methanosphaera stadtmanae is decreased. In yet anotherembodiment, the presence of a combination of archaeon genera or speciesis decreased. By way of non-limiting example, the presence ofMethanobrevibacter smithii and Methanosphaera stadtmanae is decreased.

To decrease the presence of any of the archaeon detailed above, methodsgenerally known in the art may be utilized. In one embodiment, acompound having anti-microbial activities against the archaeon isadministered to the subject. Non-limiting examples of suitableanti-microbial compounds include metronidzaole, clindamycin, timidazole,macrolides, and fluoroquinolones.

In another embodiment, a compound that inhibits methanogenesis by thearchaeon is administered to the subject. Non-limiting examples include2-bromoethanesulfonate (inhibitor of methyl-coenzyme M reductase),N-alkyl derivatives of para-aminobenzoic acid (inhibitor oftetrahydromethanopterin biosynthesis), ionophore monensin, nitroethane,lumazine, propynoic acid and ethyl 2-butynoate. In yet anotherembodiment, a hydroxymethylglutaryl-CoA reductase inhibitor isadministered to the subject. Non-limiting examples of suitablehydroxymethylglutaryl-CoA reductase inhibitors include lovastatin,atorvastatin, fluvastatin, pravastatin, simvastatin, and rosuvastatin.Alternatively, the diet of the subject may be formulated by changing thecomposition of glycans (e.g., polyfructose-containing oligosaccharides)in the diet that are preferred by polysaccharide degrading bacterialcomponents of the microbiota (e.g., Bacteroides spp) when in thepresence of mesophilic methanogenic archaeal species such asMethanobrevibacter smithii.

Generally speaking, when the archaeon population in the subject'sgastrointestinal tract is decreased in accordance with the methodsdescribed above, the polysaccharide degrading properties of thesubject's gastrointestinal microbiota is altered such thatmicrobial-mediated carbohydrate metabolism or its efficiency isdecreased. Typically, depending upon the embodiment, the transcriptomeand the metabolome of the gastrointestinal microbiota is altered. In oneembodiment, the microbe is a saccharolytic bacterium. In one alternativeof this embodiment, the saccharolytic bacterium is a Bacteroidesspecies. In a further alternative embodiment, the bacterium isBacteroides thetaiotaomicron. Typically, the carbohydrate will be aplant polysaccharide or dietary fiber. Plant polysaccharides includestarch, fructan, cellulose, hemicellulose, and pectin.

The compounds utilized in this invention to alter the archaeonpopulation may be administered by any number of routes including, butnot limited to, oral, intravenous, intramuscular, intra-arterial,intramedullary, intrathecal, intraventricular, pulmonary, transdermal,subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual,or rectal means.

The actual effective amounts of compound described herein can and willvary according to the specific composition being utilized, the mode ofadministration and the age, weight and condition of the subject. Dosagesfor a particular individual subject can be determined by one of ordinaryskill in the art using conventional considerations. Those skilled in theart will appreciate that dosages may also be determined with guidancefrom Goodman & Gilman's The Pharmacological Basis of Therapeutics, NinthEdition (1996), Appendix II, pp. 1707-1711 and from Goodman & Gilman'sThe Pharmacological Basis of Therapeutics, Tenth Edition (2001),Appendix II, pp. 475-493.

II. Biomarkers Comprising the Gut Microbiome

Another aspect of the invention encompasses use of the gut microbiome asa biomarker for obesity. The biomarker may be utilized to constructarrays that may be used for several applications including as adiagnostic or prognostic tool to determine obesity risk, judgingefficacy of existing weightloss regimes, drug discovery, for theidentification of additional biomarkers involved in obesity or anobesity related disorder, and for the discovery of therapeutic targetsinvolved in the regulation of energy balance. Generally speaking, thearray may comprise biomolecules from an obese host microbiome, includinga diet-induced obese host microbiome, or a lean host microbiome.

(a) Array

The array may be comprised of a substrate having disposed thereon atleast one biomolecule that is modulated in an obese host microbiomecompared to a lean host microbiome. Several substrates suitable for theconstruction of arrays are known in the art, and one skilled in the artwill appreciate that other substrates may become available as the artprogresses. The substrate may be a material that may be modified tocontain discrete individual sites appropriate for the attachment orassociation of the biomolecules and is amenable to at least onedetection method. Non-limiting examples of substrate materials includeglass, modified or functionalized glass, plastics (including acrylics,polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ,etc.), nylon or nitrocellulose, polysaccharides, nylon, resins, silicaor silica-based materials including silicon and modified silicon,carbon, metals, inorganic glasses and plastics. In an exemplaryembodiment, the substrates may allow optical detection withoutappreciably fluorescing.

A substrate may be planar, a substrate may be a well, i.e. a 364 wellplate, or alternatively, a substrate may be a bead. Additionally, thesubstrate may be the inner surface of a tube for flow-through sampleanalysis to minimize sample volume. Similarly, the substrate may beflexible, such as a flexible foam, including closed cell foams made ofparticular plastics.

The biomolecule or biomolecules may be attached to the substrate in awide variety of ways, as will be appreciated by those in the art. Thebiomolecule may either be synthesized first, with subsequent attachmentto the substrate, or may be directly synthesized on the substrate. Thesubstrate and the biomolecule may be derivatized with chemicalfunctional groups for subsequent attachment of the two. For example, thesubstrate may be derivatized with a chemical functional group including,but not limited to, amino groups, carboxyl groups, oxo groups or thiolgroups. Using these functional groups, the biomolecule may be attachedusing functional groups on the biomolecule either directly or indirectlyusing linkers.

The biomolecule may also be attached to the substrate non-covalently.For example, a biotinylated biomolecule can be prepared, which may bindto surfaces covalently coated with streptavidin, resulting inattachment. Alternatively, a biomolecule or biomolecules may besynthesized on the surface using techniques such as photopolymerizationand photolithography. Additional methods of attaching biomolecules toarrays and methods of synthesizing biomolecules on substrates are wellknown in the art, i.e. VLSIPS technology from Affymetrix (e.g., see U.S.Pat. No. 6,566,495, and Rockett and Dix, “DNA arrays: technology,options and toxicological applications,” Xenobiotica 30(2):155-177, allof which are hereby incorporated by reference in their entirety).

In one embodiment, the biomolecule or biomolecules attached to thesubstrate are located at a spatially defined address of the array.Arrays may comprise from about 1 to about several hundred thousandaddresses. In one embodiment, the array may be comprised of less than10,000 addresses. In another alternative embodiment, the array may becomprised of at least 10,000 addresses. In yet another alternativeembodiment, the array may be comprised of less than 5,000 addresses. Instill another alternative embodiment, the array may be comprised of atleast 5,000 addresses. In a further embodiment, the array may becomprised of less than 500 addresses. In yet a further embodiment, thearray may be comprised of at least 500 addresses.

A biomolecule may be represented more than once on a given array. Inother words, more than one address of an array may be comprised of thesame biomolecule. In some embodiments, two, three, or more than threeaddresses of the array may be comprised of the same biomolecule. Incertain embodiments, the array may comprise control biomolecules and/orcontrol addresses. The controls may be internal controls, positivecontrols, negative controls, or background controls.

The array may be comprised of biomolecules indicative of an obese hostmicrobiome. Alternatively, the array may be comprised of biomoleculesindicative of a lean host microbiome. A biomolecule is “indicative” ofan obese or lean microbiome if it tends to appear more often in one typeof microbiome compared to the other. Additionally, the array may becomprised of biomolecules that are modulated in the obese hostmicrobiome compared to the lean host microbiome. As used herein,“modulated” may refer to a biomolecule whose representation or activityis different in an obese host microbiome compared to a lean hostmicrobiome. For instance, modulated may refer to a biomolecule that isenriched, depleted, up-regulated, down-regulated, degraded, orstabilized in the obese host microbiome compared to a lean hostmicrobiome. In one embodiment, the array may be comprised of abiomolecule enriched in the obese host microbiome compared to the leanhost microbiome. In another embodiment, the array may be comprised of abiomolecule depleted in the obese host microbiome compared to the leanhost microbiome. In yet another embodiment, the array may be comprisedof a biomolecule up-regulated in the obese host microbiome compared tothe lean host microbiome. In still another embodiment, the array may becomprised of a biomolecule down-regulated in the obese host microbiomecompared to the lean host microbiome. In still yet another embodiment,the array may be comprised of a biomolecule degraded in the obese hostmicrobiome compared to the lean host microbiome. In an alternativeembodiment, the array may be comprised of a biomolecule stabilized inthe obese host microbiome compared to the lean host microbiome.

Generally speaking, an array of the invention may comprise at least onebiomolecule indicative or, or modulated in, an obese host microbiomecompared to a lean host microbiome. In one embodiment, the array maycomprise at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 biomoleculesindicative of, or modulated in, an obese host microbiome compared to alean host microbiome. In another embodiment, the array may comprise atleast 200, at least 300, at least 400, at least 500, or at least 600biomolecules indicative of, or modulated in, an obese host microbiomecompared to a lean host microbiome.

As used herein, “biomolecule” may refer to a nucleic acid, anoligonucleic acid, an amino acid, a peptide, a polypeptide, a protein, alipid, a metabolite, or a fragment thereof. Nucleic acids may includeRNA, DNA, and naturally occurring or synthetically created derivatives.A biomolecule may be present in, produced by, or modified by amicroorganism within the gut.

Biomolecules that are enriched in the obese microbiome compared to thelean microbiome may include biomolecules derived from the followingKyoto Encyclopedia of Genes and Genomes (KEGG) Categories: CarbohydrateMetabolism, Amino Acid Metabolism, Metabolism of Other Amino Acids,Glycan Biosynthesis and Metabolism, Biosynthesis of Polyketides andNonribosomal Peptides, Transcription, Folding/Sorting/Degradation,Signal Transduction, and Cell Growth and Death. In certain embodiments,the biomolecules derived from the KEGG categories above may includebiomolecules from a corresponding KEGG pathway (see Examples).Additionally, biomolecules that are enriched in the obese microbiomecompared to the lean microbiome may include nucleic acids encodingproteins or portions of proteins derived from the following Clusters ofOrthologous Genes (COGs): Transcription,Replication/recombination/repair, Nuclear structure, signaltransduction, cell wall/membrane/envelope biogenesis, Energy production,Nucleotide, Ion, and cell motility.

Alternatively, biomolecules that are depleted in the obese microbiomecompared to the lean microbiome may be biomolecules derived from thefollowing KEGG categories: Carbohydrate Metabolism, Energy Metabolism,Lipid Metabolism, Nucleotide Metabolism, Amino Acid Metabolism, GlycanBiosynthesis and Metabolism, Metabolism of Cofactors and Vitamins,Translation, and Folding/Sorting/Degradation. In certain embodiments,the biomolecules encoding proteins or portions of proteins derived fromthe KEGG categories above may include biomolecules from a correspondingKEGG pathway (see Examples). Additionally, biomolecules that aredepleted in the obese microbiome compared to the lean microbiome mayinclude biomolecules encoding proteins or portions of proteins derivedfrom the following COGs: Translation, Defense Mechanisms, EnergyProduction, Nucleotide, Coenzyme, Ion, and Posttranslationalmodification/protein turnover/chaperones.

Biomolecules indicative of, or modulated in, an obese host microbiomecompared to a lean host microbiome may include biomolecules associatedwith di- and poly-saccharide (fructoside) degradation, such as ‘fructanbeta-fructosidase’ (K03332), a gene that allows the degradation ofsucrose, inulin, and/or levan, or a biomolecule associated with the KEGGpathway for fructose and mannose metabolism. Additionally, the array mayinclude biomolecules associated with the import of mono- anddi-saccharides via the Phosphotransferase system (PTS), such as asbiomolecules for importing and metabolizing fructose, glucose,N-acetyl-glucosamine, and N-acetyl-galactosamine. Also, the array mayinclude biomolecules associated with the Metabolism of importedcarbohydrates, such as biomolecules associated with the KEGG pathway forGlycolysis, including biomolecules to process imported carbohydrates tophosphoenolpyruvate (PEP). The array may further include biomolecuesassociated with anaerobic fermentation, such as biomolecules associatedwith the pathways for the fermentation of carbohydrates to acetate,butyrate, and lactate. In each of the above embodiments, thebiomolecules are indicative of, or modulated in, an obese hostmicrobiome compared to a lean host microbiome.

In some embodiments, the biomolecules of the array may be selected frombiomolecules involved in polysaccharide degradation. For instance, thearray may comprise biomolecules involved in polysaccharide degradationthat are indicative of, or modulated in, an obese host microbiomecompared to a lean host microbiome. In particular, the array maycomprise glycoside hydrolases that are indicative of, or modulated in,an obese host microbiome compared to the lean host microbiome. In oneembodiment, the array may comprise biomolecules from the CAZy familes 2,4, 27, 31, 35, 36, 42, and 68 that are indicative of or modulated in anobese host microbiome compared to a lean host microbiome. In anotherembodiment, the array may comprise biomolecules from the CAZy families2, 4, 27, 31, 35, 36, 42, and 68 that are up-regulated or enriched in anobese host microbiome compared to a lean host microbiome. The CAZydatabase describes the families of structurally-related catalytic andcarbohydrate-binding modules (or functional domains) of enzymes thatdegrade, modify, or create glycosidic bonds, and may be accessed atwww.cazy.org/index.html. In another embodiment, the array may comprisealpha-galactosidases, beta-galactosidases, alpha-amylases andamylomaltases that are indicative of, or modulated in, an obese hostmicrobiome compared to a lean host microbiome. Additionally, the arraymay comprise biomolecules selected from the KEGG pathways for starch andsucrose metabolism, galactose metabolism, and butanoate metabolism thatare indicative of, or modulated in, an obese host microbiome compared toa lean host microbiome (See Tables Z, Y, and X).

In other embodiments, the biomolecules of the array may be selected frombiomolecules involved in carbohydrate import that are indicative of, ormodulated in, an obese host microbiome compared to a lean hostmicrobiome. For instance, the biomolecules may be ABC transporters (SeeTable V). In yet another embodiment, the biomolecules may be selectedfrom biomolecules involved in acetogenesis, or the generation of acetatefrom CO₂ (See Table W). For instance, the biomolecule may be aformate-tetrahydrofolate ligase.

In still other embodiments, the biomolecules may be selected frombiomolecules involved in anaerobic fermentation that are indicative of,or modulated in, an obese host microbiome compared to a lean hostmicrobiome. For instance, the biomolecules may be selected frombiomolecules involved in the fermentation of carbohydrates to acetateand butyrate. Specifically, the biomarker may comprise pyruvateformate-lyase. Alternatively, the biomarker may comprise biomolecules inthe KEGG butanoate metabolism pathway (See Table X).

In certain embodiments, the biomolecules of the array may be selectedfrom the nucleic acid sequences represented by GenBank project accessionnumbers AATA00000000-AATF00000000, i.e. including the AATB, AATC, AATD,and AATE accession numbers. Alternatively, the biomolecules may beselected from the proteins encoded by the nucleic acid sequencesrepresented by GenBank project accession numbersAATA00000000-AATF00000000, i.e. including the AATB, AATC, AATD, and AATEaccession numbers. In some embodiments, the biomolecules may be selectedfrom the nucleic acid sequences represented by GenBank project accessionnumbers AATA00000000-AATF00000000, i.e. including the AATB, AATC, AATD,and AATE accession numbers that are modulated in the obese hostmicrobiome compared to the lean host microbiome. In another alternative,the biomolecules may be selected from the proteins encoded by thenucleic acid sequences represented by GenBank project accession numbersAATA00000000-AATF00000000, i.e. including the AATB, AATC, AATD, and AATEaccession numbers that are modulated in the obese host microbiomecompared to the lean host microbiome.

In several embodiments, the biomolecules of the array may be selectedfrom the biomolecules represented by the accession numbers listed inTables Z-V. Table Z represents the accession numbers of 629 biomoleculesinvolved in starch and sucrose metabolism that are enriched in the obesehost microbiome compared to the lean host microbiome. Table Y representsthe accession numbers of 205 biomolecules involved in galactosemetabolism that are enriched in the obese host microbiome compared tothe lean host microbiome. Table X represents the accession numbers of124 biomolecules involved in butanoate metabolism that are enriched inthe obese host microbiome compared to the lean host microbiome. Table Wrepresents the accession numbers of 14 biomolecules involved inacetogenesis that are enriched in the obese host microbiome compared tothe lean host microbiome. Table V represents the accession numbers of869 biomolecules involved in carbohydrate import that are enriched inthe obese host microbiome compared to the lean host microbiome.

Additionally, the biomolecule may be at least 70, 75, 80, 85, 90, or 95%homologous to a biomolecule derived from an accession number detailedabove. In one embodiment, the biomolecule may be at least 80, 81, 82,83, 84, 85, 86, 87, 88, or 89% homologous to a biomolecule derived froman accession number detailed above. In another embodiment, thebiomolecule may be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%homologous to a biomolecule derived from an accession number detailedabove.

In determining whether a biomolecule is substantially homologous orshares a certain percentage of sequence identity with a sequence of theinvention, sequence similarity may be determined by conventionalalgorithms, which typically allow introduction of a small number of gapsin order to achieve the best fit. In particular, “percent identity” oftwo polypeptides or two nucleic acid sequences is determined using thealgorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA87:2264-2268, 1993). Such an algorithm is incorporated into the BLASTNand BLASTX programs of Altschul et al. (J. Mol. Biol. 215:403-410,1990). BLAST nucleotide searches may be performed with the BLASTNprogram to obtain nucleotide sequences homologous to a nucleic acidmolecule of the invention. Equally, BLAST protein searches may beperformed with the BLASTX program to obtain amino acid sequences thatare homologous to a polypeptide of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST is utilized asdescribed in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., BLASTX and BLASTN) are employed. Seewww.ncbi.nlm.nih.gov for more details.

For each of the above embodiments, methods of determining biomoleculesthat are indicative or, or modulated in, an obese host microbiomecompared to a lean host microbiome may be determined using methodsdetailed in the Examples.

TABLE Z Starch and sucrose metabolism AATA01000367.1 AATA01000378.1AATA01000604.1 AATA01000619.1 AATA01000626.1 AATA01000812.1AATA01000861.1 AATA01001081.1 AATA01001162.1 AATA01001279.1AATA01001315.1 AATA01001352.1 AATA01001552.1 AATA01001626.1AATA01001645.1 AATA01001835.1 AATA01001927.1 AATA01002235.1AATA01002243.1 AATA01002245.1 AATA01002354.1 AATA01002406.1AATA01002523.1 AATA01002663.1 AATA01002708.1 AATA01002712.1AATA01002826.1 AATA01002865.1 AATA01002884.1 AATA01002939.1AATA01002955.1 AATA01002994.1 AATA01003014.1 AATA01003144.1AATA01003220.1 AATA01003314.1 AATA01003592.1 AATA01003657.1AATA01003741.1 AATA01003877.1 AATA01004137.1 AATA01004174.1AATA01004366.1 AATA01004387.1 AATA01004465.1 AATA01004518.1AATA01004607.1 AATA01004681.1 AATA01004688.1 AATA01004723.1AATA01004736.1 AATA01004871.1 AATA01004904.1 AATA01004932.1AATA01005085.1 AATA01005122.1 AATA01005201.1 AATA01005300.1AATA01005319.1 AATA01005501.1 AATA01005538.1 AATA01005692.1AATA01005728.1 AATA01006002.1 AATA01006129.1 AATA01006149.1AATA01006278.1 AATA01006286.1 AATA01006335.1 AATA01006431.1AATA01006513.1 AATA01006879.1 AATA01006985.1 AATA01007186.1AATA01007348.1 AATA01007637.1 AATA01007781.1 AATA01008402.1AATA01008580.1 AATA01008670.1 AATA01008918.1 AATA01009070.1AATA01009141.1 AATA01009144.1 AATA01009153.1 AATA01009379.1AATA01009421.1 AATA01009439.1 AATA01009527.1 AATA01009635.1AATA01009673.1 AATA01009760.1 AATA01009879.1 AATA01010032.1AATA01010127.1 AATA01010306.1 AATA01010423.1 AATB01000541.1AATB01000828.1 AATB01000866.1 AATB01001143.1 AATB01001302.1AATB01001307.1 AATB01001311.1 AATB01001359.1 AATB01001422.1AATB01001587.1 AATB01001641.1 AATB01001707.1 AATB01001953.1AATB01001986.1 AATB01001991.1 AATB01002005.1 AATB01002085.1AATB01002213.1 AATB01002368.1 AATB01002391.1 AATB01002599.1AATB01002717.1 AATB01002889.1 AATB01002892.1 AATB01003163.1AATB01003217.1 AATB01003396.1 AATB01003515.1 AATB01003596.1AATB01003671.1 AATB01003715.1 AATB01003838.1 AATB01003979.1AATB01004028.1 AATB01004958.1 AATB01005356.1 AATB01006000.1AATB01006136.1 AATB01006159.1 AATB01006233.1 AATB01006389.1AATB01006647.1 AATB01006754.1 AATB01006908.1 AATB01006919.1AATB01006926.1 AATB01006935.1 AATB01007750.1 AATB01007943.1AATB01008155.1 AATB01008432.1 AATB01008453.1 AATB01008635.1AATB01008636.1 AATB01009265.1 AATB01009430.1 AATB01009668.1AATB01009708.1 AATB01009907.1 AATB01009949.1 AATB01010152.1AATB01010429.1 AATB01010485.1 AATB01010566.1 AATB01010591.1AATB01010614.1 AATB01010703.1 AATB01011114.1 AATB01011135.1AATC01000258.1 AATC01000287.1 AATC01000304.1 AATC01000371.1AATC01000530.1 AATC01000565.1 AATC01000603.1 AATC01000608.1AATC01000684.1 AATC01000687.1 AATC01000731.1 AATC01000774.1AATC01000842.1 AATC01000892.1 AATC01000911.1 AATC01000998.1AATC01001054.1 AATC01001100.1 AATC01001151.1 AATC01001177.1AATC01001184.1 AATC01001227.1 AATC01001267.1 AATC01001350.1AATC01001408.1 AATC01001426.1 AATC01001459.1 AATC01001480.1AATC01001552.1 AATC01001685.1 AATC01001711.1 AATC01001747.1AATC01001759.1 AATC01001846.1 AATC01001990.1 AATC01002000.1AATC01002009.1 AATC01002024.1 AATC01002090.1 AATC01002127.1AATC01002172.1 AATC01002210.1 AATC01002299.1 AATC01002329.1AATC01002344.1 AATC01002357.1 AATC01002445.1 AATC01002465.1AATC01002560.1 AATC01002578.1 AATC01002607.1 AATC01002624.1AATC01002648.1 AATC01002727.1 AATC01002866.1 AATC01002879.1AATC01002942.1 AATC01002955.1 AATC01002963.1 AATC01003024.1AATC01003029.1 AATC01003130.1 AATC01003154.1 AATC01003160.1AATC01003195.1 AATC01003200.1 AATC01003225.1 AATC01003262.1AATC01003382.1 AATC01003392.1 AATC01003402.1 AATC01003434.1AATC01003436.1 AATC01003568.1 AATC01003650.1 AATC01003652.1AATC01003716.1 AATC01003871.1 AATC01003874.1 AATC01003891.1AATC01003916.1 AATC01003933.1 AATC01003992.1 AATC01004072.1AATC01004086.1 AATC01004275.1 AATC01004294.1 AATC01004330.1AATC01004346.1 AATC01004392.1 AATC01004398.1 AATC01004407.1AATC01004442.1 AATC01004563.1 AATC01004594.1 AATC01004622.1AATC01004683.1 AATC01004784.1 AATC01004844.1 AATC01004885.1AATC01004949.1 AATC01004952.1 AATC01004959.1 AATC01004979.1AATC01004992.1 AATC01005038.1 AATC01005060.1 AATC01005067.1AATC01005139.1 AATC01005150.1 AATC01005255.1 AATC01005305.1AATC01005366.1 AATC01005548.1 AATC01005596.1 AATC01005667.1AATC01005710.1 AATC01005725.1 AATC01005781.1 AATC01005791.1AATC01005825.1 AATC01005892.1 AATC01005918.1 AATC01005994.1AATC01006282.1 AATC01006321.1 AATC01006345.1 AATC01006348.1AATC01006547.1 AATC01006642.1 AATC01006644.1 AATC01006710.1AATC01006770.1 AATC01006788.1 AATC01006794.1 AATC01006798.1AATC01006801.1 AATC01006817.1 AATC01006895.1 AATC01006950.1AATC01006976.1 AATC01007020.1 AATC01007041.1 AATC01007109.1AATC01007158.1 AATC01007175.1 AATC01007207.1 AATC01007233.1AATC01007273.1 AATC01007507.1 AATC01007551.1 AATC01007571.1AATC01007583.1 AATC01007608.1 AATC01007644.1 AATC01007696.1AATC01007715.1 AATC01007756.1 AATC01007782.1 AATC01007812.1AATC01007882.1 AATC01007944.1 AATC01008034.1 AATC01008188.1AATC01008195.1 AATC01008305.1 AATC01008420.1 AATC01008484.1AATC01008559.1 AATC01008756.1 AATC01008968.1 AATC01008973.1AATC01009076.1 AATC01009132.1 AATC01009148.1 AATC01009210.1AATD01000417.1 AATD01000450.1 AATD01000483.1 AATD01000495.1AATD01000578.1 AATD01000590.1 AATD01000592.1 AATD01000617.1AATD01000667.1 AATD01000692.1 AATD01000700.1 AATD01000701.1AATD01000817.1 AATD01000870.1 AATD01000895.1 AATD01001162.1AATD01001216.1 AATD01001248.1 AATD01001259.1 AATD01001305.1AATD01001317.1 AATD01001322.1 AATD01001360.1 AATD01001400.1AATD01001534.1 AATD01001567.1 AATD01001580.1 AATD01001592.1AATD01001653.1 AATD01001690.1 AATD01001755.1 AATD01001774.1AATD01001896.1 AATD01001902.1 AATD01001918.1 AATD01001922.1AATD01001948.1 AATD01001974.1 AATD01002028.1 AATD01002041.1AATD01002051.1 AATD01002095.1 AATD01002097.1 AATD01002126.1AATD01002165.1 AATD01002168.1 AATD01002169.1 AATD01002186.1AATD01002395.1 AATD01002453.1 AATD01002472.1 AATD01002548.1AATD01002596.1 AATD01002739.1 AATD01002778.1 AATD01002817.1AATD01002964.1 AATD01003003.1 AATD01003182.1 AATD01003199.1AATD01003222.1 AATD01003264.1 AATD01003296.1 AATD01003384.1AATD01003453.1 AATD01003557.1 AATD01003608.1 AATD01003759.1AATD01003803.1 AATD01003884.1 AATD01004067.1 AATD01004083.1AATD01004161.1 AATD01004184.1 AATD01004186.1 AATD01004300.1AATD01004313.1 AATD01004319.1 AATD01004475.1 AATD01004607.1AATD01004618.1 AATD01004644.1 AATD01004760.1 AATD01004779.1AATD01004788.1 AATD01004797.1 AATD01004923.1 AATD01004935.1AATD01004970.1 AATD01005048.1 AATD01005176.1 AATD01005198.1AATD01005260.1 AATD01005276.1 AATD01005402.1 AATD01005457.1AATD01005559.1 AATD01005574.1 AATD01005580.1 AATD01005613.1AATD01005675.1 AATD01005694.1 AATD01005742.1 AATD01005743.1AATD01005837.1 AATD01005915.1 AATD01005919.1 AATD01005940.1AATD01005958.1 AATD01005992.1 AATD01006063.1 AATD01006088.1AATD01006123.1 AATD01006191.1 AATD01006205.1 AATD01006240.1AATD01006275.1 AATD01006409.1 AATD01006524.1 AATD01006610.1AATD01006638.1 AATD01006719.1 AATD01006732.1 AATD01006783.1AATD01007055.1 AATD01007082.1 AATD01007119.1 AATD01007171.1AATD01007291.1 AATD01007301.1 AATD01007386.1 AATD01007431.1AATD01007525.1 AATD01007572.1 AATD01007645.1 AATD01007670.1AATD01007680.1 AATD01007739.1 AATD01007740.1 AATD01007760.1AATD01007763.1 AATD01007884.1 AATD01007984.1 AATD01008070.1AATD01008133.1 AATD01008140.1 AATD01008333.1 AATD01008354.1AATD01008358.1 AATD01008447.1 AATD01008482.1 AATD01008755.1AATD01008814.1 AATD01008829.1 AATD01008904.1 AATD01008967.1AATD01009012.1 AATD01009079.1 AATD01009091.1 AATD01009209.1AATD01009218.1 AATD01009406.1 AATD01009708.1 AATD01009803.1AATD01009887.1 AATD01010045.1 AATD01010117.1 AATD01010291.1AATD01010417.1 AATE01000308.1 AATE01000370.1 AATE01000448.1AATE01000480.1 AATE01000499.1 AATE01000507.1 AATE01000582.1AATE01000587.1 AATE01000694.1 AATE01000769.1 AATE01000944.1AATE01001080.1 AATE01001116.1 AATE01001133.1 AATE01001191.1AATE01001255.1 AATE01001284.1 AATE01001287.1 AATE01001291.1AATE01001296.1 AATE01001322.1 AATE01001391.1 AATE01001410.1AATE01001429.1 AATE01001447.1 AATE01001485.1 AATE01001571.1AATE01001605.1 AATE01001726.1 AATE01001837.1 AATE01001916.1AATE01002002.1 AATE01002010.1 AATE01002054.1 AATE01002129.1AATE01002478.1 AATE01002491.1 AATE01002639.1 AATE01002642.1AATE01002752.1 AATE01002805.1 AATE01002827.1 AATE01002876.1AATE01002910.1 AATE01002927.1 AATE01002930.1 AATE01002966.1AATE01003068.1 AATE01003115.1 AATE01003117.1 AATE01003209.1AATE01003321.1 AATE01003471.1 AATE01003513.1 AATE01003545.1AATE01003606.1 AATE01003640.1 AATE01003711.1 AATE01003753.1AATE01003797.1 AATE01003918.1 AATE01003988.1 AATE01004230.1AATE01004265.1 AATE01004275.1 AATE01004341.1 AATE01004344.1AATE01004359.1 AATE01004397.1 AATE01004780.1 AATE01004806.1AATE01004832.1 AATE01004848.1 AATE01004874.1 AATE01005032.1AATE01005110.1 AATE01005223.1 AATE01005284.1 AATE01005347.1AATE01005425.1 AATE01005430.1 AATE01005453.1 AATE01005503.1AATE01005516.1 AATE01005628.1 AATE01005751.1 AATE01005984.1AATE01005987.1 AATE01005997.1 AATE01006310.1 AATE01006483.1AATE01006505.1 AATE01006523.1 AATE01006684.1 AATE01006715.1AATE01006774.1 AATE01006838.1 AATE01006921.1 AATE01006965.1AATE01006992.1 AATE01007020.1 AATE01007104.1 AATE01007332.1AATE01007446.1 AATE01007477.1 AATE01007487.1 AATE01007572.1AATE01007581.1 AATE01007637.1 AATE01007670.1 AATE01007813.1AATE01007853.1 AATE01007863.1 AATE01007865.1 AATE01008009.1AATE01008113.1 AATE01008332.1 AATE01008416.1

TABLE Y Galactose metabolism AATA01000364.1 AATA01001208.1AATA01001269.1 AATA01001302.1 AATA01001530.1 AATA01001794.1AATA01001880.1 AATA01001927.1 AATA01001998.1 AATA01002782.1AATA01002826.1 AATA01002838.1 AATA01002927.1 AATA01003314.1AATA01003511.1 AATA01003657.1 AATA01004057.1 AATA01004156.1AATA01004179.1 AATA01004301.1 AATA01004387.1 AATA01004448.1AATA01004634.1 AATA01004643.1 AATA01004657.1 AATA01004683.1AATA01005518.1 AATA01005535.1 AATA01006014.1 AATA01006041.1AATA01006173.1 AATA01006335.1 AATA01006349.1 AATA01006704.1AATA01007290.1 AATA01007352.1 AATA01007470.1 AATA01007717.1AATA01008261.1 AATA01008580.1 AATA01008582.1 AATA01008670.1AATA01008996.1 AATA01009120.1 AATA01009155.1 AATA01009419.1AATA01009690.1 AATA01009869.1 AATA01009914.1 AATA01009942.1AATA01010032.1 AATA01010051.1 AATB01000866.1 AATB01000983.1AATB01001125.1 AATB01002005.1 AATB01002085.1 AATB01002128.1AATB01002512.1 AATB01002942.1 AATB01003728.1 AATB01004292.1AATB01004589.1 AATB01004893.1 AATB01005776.1 AATB01005876.1AATB01006159.1 AATB01006233.1 AATB01006707.1 AATB01006981.1AATB01007416.1 AATB01007666.1 AATB01008155.1 AATB01008668.1AATB01009265.1 AATB01009587.1 AATB01009693.1 AATB01009765.1AATB01010238.1 AATB01010566.1 AATB01010624.1 AATC01000464.1AATC01000511.1 AATC01000579.1 AATC01000949.1 AATC01001846.1AATC01001944.1 AATC01002423.1 AATC01002942.1 AATC01003054.1AATC01003114.1 AATC01003382.1 AATC01003568.1 AATC01003750.1AATC01004209.1 AATC01005013.1 AATC01005150.1 AATC01005251.1AATC01005327.1 AATC01005335.1 AATC01005489.1 AATC01005624.1AATC01005791.1 AATC01005825.1 AATC01005918.1 AATC01005978.1AATC01006168.1 AATC01006305.1 AATC01006895.1 AATC01007014.1AATC01007273.1 AATC01007447.1 AATC01007620.1 AATC01007699.1AATC01007715.1 AATC01007759.1 AATC01007944.1 AATC01008188.1AATC01008273.1 AATC01009076.1 AATC01009132.1 AATC01009381.1AATC01009482.1 AATC01009752.1 AATD01000574.1 AATD01000948.1AATD01000982.1 AATD01001338.1 AATD01001342.1 AATD01001360.1AATD01001567.1 AATD01002333.1 AATD01002469.1 AATD01002969.1AATD01003167.1 AATD01003676.1 AATD01003784.1 AATD01003919.1AATD01004004.1 AATD01004357.1 AATD01004715.1 AATD01004791.1AATD01004845.1 AATD01004887.1 AATD01005117.1 AATD01005494.1AATD01005874.1 AATD01006550.1 AATD01006577.1 AATD01006585.1AATD01007211.1 AATD01007618.1 AATD01007837.1 AATD01007984.1AATD01008181.1 AATD01008191.1 AATD01008355.1 AATD01008641.1AATD01008755.1 AATD01009058.1 AATD01009102.1 AATD01009377.1AATD01009406.1 AATD01009509.1 AATD01009708.1 AATD01010045.1AATD01010150.1 AATD01010417.1 AATE01000573.1 AATE01000685.1AATE01000743.1 AATE01001204.1 AATE01001517.1 AATE01001588.1AATE01001661.1 AATE01001729.1 AATE01001735.1 AATE01001837.1AATE01001859.1 AATE01001929.1 AATE01001932.1 AATE01002180.1AATE01002491.1 AATE01002500.1 AATE01002777.1 AATE01002846.1AATE01003919.1 AATE01004109.1 AATE01004230.1 AATE01004342.1AATE01004487.1 AATE01004600.1 AATE01004792.1 AATE01004793.1AATE01005455.1 AATE01005465.1 AATE01005628.1 AATE01005987.1AATE01006089.1 AATE01006333.1 AATE01006472.1 AATE01007195.1AATE01007261.1 AATE01007301.1 AATE01007912.1

TABLE X Butanoate metabolism AATA01000644.1 AATA01001167.1AATA01001250.1 AATA01002159.1 AATA01002922.1 AATA01003720.1AATA01003830.1 AATA01004132.1 AATA01004146.1 AATA01004278.1AATA01004287.1 AATA01004779.1 AATA01005204.1 AATA01005614.1AATA01006915.1 AATA01008164.1 AATA01009218.1 AATA01009505.1AATA01009533.1 AATA01009725.1 AATA01010088.1 AATA01010256.1AATB01000530.1 AATB01000821.1 AATB01003115.1 AATB01003466.1AATB01003612.1 AATB01003692.1 AATB01003748.1 AATB01004113.1AATB01005179.1 AATB01005626.1 AATB01006406.1 AATB01007003.1AATB01007143.1 AATB01007347.1 AATB01007536.1 AATB01007719.1AATB01009516.1 AATB01010198.1 AATB01010413.1 AATB01010772.1AATC01000930.1 AATC01001211.1 AATC01001417.1 AATC01001542.1AATC01001583.1 AATC01001785.1 AATC01002540.1 AATC01003252.1AATC01003508.1 AATC01003890.1 AATC01004159.1 AATC01004206.1AATC01004856.1 AATC01005074.1 AATC01005740.1 AATC01006325.1AATC01006593.1 AATC01006610.1 AATC01007057.1 AATC01007281.1AATC01007335.1 AATC01007438.1 AATC01007575.1 AATC01007815.1AATC01008204.1 AATC01008348.1 AATC01008417.1 AATC01008488.1AATC01008728.1 AATC01009201.1 AATC01009321.1 AATC01009428.1AATD01000560.1 AATD01001080.1 AATD01001118.1 AATD01001861.1AATD01002172.1 AATD01002433.1 AATD01002839.1 AATD01003082.1AATD01003422.1 AATD01003433.1 AATD01003491.1 AATD01004323.1AATD01006189.1 AATD01007344.1 AATD01007960.1 AATD01007980.1AATD01007981.1 AATD01008180.1 AATD01008255.1 AATD01008286.1AATD01009476.1 AATD01009477.1 AATD01009533.1 AATD01009692.1AATD01009926.1 AATD01009946.1 AATD01010353.1 AATE01000586.1AATE01001750.1 AATE01002442.1 AATE01002516.1 AATE01002768.1AATE01002862.1 AATE01003029.1 AATE01003071.1 AATE01003308.1AATE01003617.1 AATE01003652.1 AATE01004436.1 AATE01004528.1AATE01004575.1 AATE01005584.1 AATE01005838.1 AATE01006057.1AATE01006232.1 AATE01006597.1 AATE01007131.1 AATE01007812.1AATE01007939.1 AATE01008055.1

TABLE W Acetogenesis AATA01001505.1 AATA01004859.1 AATB01006849.1AATC01008727.1 AATC01009120.1 AATD01002372.1 AATD01008617.1AATD01008638.1 AATD01010214.1 AATD01010397.1 AATE01002545.1AATE01003350.1 AATE01006283.1 AATE01006955.1

TABLE V Carbohydrate import AATA01000244.1 AATA01000256.1 AATA01000264.1AATA01000407.1 AATA01000454.1 AATA01000460.1 AATA01000649.1AATA01000657.1 AATA01000704.1 AATA01000718.1 AATA01000840.1AATA01000914.1 AATA01000918.1 AATA01000924.1 AATA01000941.1AATA01000948.1 AATA01001055.1 AATA01001105.1 AATA01001107.1AATA01001118.1 AATA01001169.1 AATA01001184.1 AATA01001211.1AATA01001240.1 AATA01001350.1 AATA01001402.1 AATA01001449.1AATA01001468.1 AATA01001519.1 AATA01001548.1 AATA01001596.1AATA01001674.1 AATA01001683.1 AATA01001756.1 AATA01001798.1AATA01001826.1 AATA01001960.1 AATA01001988.1 AATA01002023.1AATA01002061.1 AATA01002091.1 AATA01002097.1 AATA01002127.1AATA01002286.1 AATA01002295.1 AATA01002302.1 AATA01002314.1AATA01002315.1 AATA01002485.1 AATA01002533.1 AATA01002571.1AATA01002592.1 AATA01002623.1 AATA01002702.1 AATA01002745.1AATA01002806.1 AATA01002830.1 AATA01003217.1 AATA01003262.1AATA01003344.1 AATA01003372.1 AATA01003398.1 AATA01003463.1AATA01003589.1 AATA01003600.1 AATA01003629.1 AATA01003690.1AATA01003817.1 AATA01003835.1 AATA01003903.1 AATA01003919.1AATA01003978.1 AATA01003984.1 AATA01004149.1 AATA01004209.1AATA01004273.1 AATA01004289.1 AATA01004378.1 AATA01004450.1AATA01004524.1 AATA01004807.1 AATA01004870.1 AATA01004892.1AATA01004901.1 AATA01004924.1 AATA01005026.1 AATA01005173.1AATA01005175.1 AATA01005188.1 AATA01005375.1 AATA01005474.1AATA01005476.1 AATA01005513.1 AATA01005542.1 AATA01005605.1AATA01005621.1 AATA01005635.1 AATA01005718.1 AATA01005737.1AATA01005795.1 AATA01005832.1 AATA01005849.1 AATA01005865.1AATA01006008.1 AATA01006060.1 AATA01006125.1 AATA01006136.1AATA01006198.1 AATA01006210.1 AATA01006289.1 AATA01006308.1AATA01006357.1 AATA01006404.1 AATA01006447.1 AATA01006466.1AATA01006496.1 AATA01006517.1 AATA01006537.1 AATA01006561.1AATA01006573.1 AATA01006591.1 AATA01006676.1 AATA01006731.1AATA01006792.1 AATA01006823.1 AATA01006839.1 AATA01006863.1AATA01006887.1 AATA01006917.1 AATA01006940.1 AATA01006964.1AATA01007124.1 AATA01007141.1 AATA01007312.1 AATA01007314.1AATA01007357.1 AATA01007369.1 AATA01007395.1 AATA01007422.1AATA01007430.1 AATA01007488.1 AATA01007553.1 AATA01007571.1AATA01007610.1 AATA01007648.1 AATA01007651.1 AATA01007792.1AATA01007869.1 AATA01007892.1 AATA01007900.1 AATA01008013.1AATA01008049.1 AATA01008168.1 AATA01008203.1 AATA01008257.1AATA01008283.1 AATA01008296.1 AATA01008314.1 AATA01008323.1AATA01008515.1 AATA01008573.1 AATA01008611.1 AATA01008856.1AATA01008860.1 AATA01008926.1 AATA01009002.1 AATA01009048.1AATA01009079.1 AATA01009208.1 AATA01009214.1 AATA01009245.1AATA01009367.1 AATA01009381.1 AATA01009437.1 AATA01009778.1AATA01009988.1 AATA01010002.1 AATA01010010.1 AATA01010140.1AATA01010160.1 AATA01010161.1 AATA01010246.1 AATA01010254.1AATA01010284.1 AATA01010321.1 AATA01010426.1 AATA01010491.1AATB01000575.1 AATB01000581.1 AATB01000602.1 AATB01000722.1AATB01000851.1 AATB01000872.1 AATB01000880.1 AATB01000886.1AATB01000919.1 AATB01000970.1 AATB01001009.1 AATB01001158.1AATB01001176.1 AATB01001186.1 AATB01001343.1 AATB01001385.1AATB01001522.1 AATB01001564.1 AATB01001581.1 AATB01001630.1AATB01001748.1 AATB01001765.1 AATB01001951.1 AATB01001983.1AATB01002020.1 AATB01002029.1 AATB01002033.1 AATB01002052.1AATB01002107.1 AATB01002121.1 AATB01002216.1 AATB01002266.1AATB01002400.1 AATB01002415.1 AATB01002469.1 AATB01002485.1AATB01002514.1 AATB01002749.1 AATB01003053.1 AATB01003184.1AATB01003196.1 AATB01003215.1 AATB01003233.1 AATB01003278.1AATB01003643.1 AATB01003900.1 AATB01003909.1 AATB01004000.1AATB01004086.1 AATB01004172.1 AATB01004341.1 AATB01004438.1AATB01004470.1 AATB01004487.1 AATB01004670.1 AATB01004797.1AATB01004850.1 AATB01004902.1 AATB01005206.1 AATB01005283.1AATB01005386.1 AATB01005444.1 AATB01005574.1 AATB01005614.1AATB01005733.1 AATB01005987.1 AATB01006042.1 AATB01006334.1AATB01006560.1 AATB01006677.1 AATB01006739.1 AATB01006907.1AATB01007087.1 AATB01007199.1 AATB01007305.1 AATB01007439.1AATB01007624.1 AATB01007758.1 AATB01007777.1 AATB01007842.1AATB01008029.1 AATB01008092.1 AATB01008115.1 AATB01008159.1AATB01008190.1 AATB01008378.1 AATB01008550.1 AATB01008589.1AATB01008901.1 AATB01008990.1 AATB01009020.1 AATB01009175.1AATB01009262.1 AATB01009270.1 AATB01009476.1 AATB01009482.1AATB01009498.1 AATB01009508.1 AATB01009546.1 AATB01009832.1AATB01009964.1 AATB01010358.1 AATB01010453.1 AATB01010547.1AATB01010598.1 AATB01010794.1 AATB01010980.1 AATB01011205.1AATC01000266.1 AATC01000276.1 AATC01000400.1 AATC01000444.1AATC01000469.1 AATC01000480.1 AATC01000529.1 AATC01000552.1AATC01000557.1 AATC01000633.1 AATC01000641.1 AATC01000672.1AATC01000699.1 AATC01000706.1 AATC01000793.1 AATC01000952.1AATC01001028.1 AATC01001040.1 AATC01001103.1 AATC01001127.1AATC01001141.1 AATC01001370.1 AATC01001394.1 AATC01001427.1AATC01001431.1 AATC01001570.1 AATC01001618.1 AATC01001666.1AATC01001673.1 AATC01001734.1 AATC01001739.1 AATC01001766.1AATC01001783.1 AATC01001852.1 AATC01001873.1 AATC01001946.1AATC01001947.1 AATC01002005.1 AATC01002016.1 AATC01002057.1AATC01002196.1 AATC01002260.1 AATC01002281.1 AATC01002369.1AATC01002400.1 AATC01002415.1 AATC01002436.1 AATC01002700.1AATC01002709.1 AATC01002819.1 AATC01002959.1 AATC01003071.1AATC01003221.1 AATC01003254.1 AATC01003343.1 AATC01003349.1AATC01003428.1 AATC01003517.1 AATC01003579.1 AATC01003690.1AATC01003703.1 AATC01003794.1 AATC01003818.1 AATC01003838.1AATC01003842.1 AATC01003886.1 AATC01004096.1 AATC01004140.1AATC01004146.1 AATC01004189.1 AATC01004263.1 AATC01004439.1AATC01004488.1 AATC01004536.1 AATC01004580.1 AATC01004627.1AATC01004739.1 AATC01004818.1 AATC01005066.1 AATC01005092.1AATC01005108.1 AATC01005125.1 AATC01005175.1 AATC01005254.1AATC01005273.1 AATC01005326.1 AATC01005328.1 AATC01005372.1AATC01005390.1 AATC01005427.1 AATC01005464.1 AATC01005470.1AATC01005513.1 AATC01005583.1 AATC01005702.1 AATC01005707.1AATC01005738.1 AATC01005742.1 AATC01005875.1 AATC01005876.1AATC01006042.1 AATC01006133.1 AATC01006148.1 AATC01006231.1AATC01006314.1 AATC01006341.1 AATC01006364.1 AATC01006389.1AATC01006444.1 AATC01006537.1 AATC01006546.1 AATC01006554.1AATC01006560.1 AATC01006608.1 AATC01006664.1 AATC01006925.1AATC01007029.1 AATC01007324.1 AATC01007358.1 AATC01007560.1AATC01007857.1 AATC01007881.1 AATC01008013.1 AATC01008032.1AATC01008115.1 AATC01008201.1 AATC01008216.1 AATC01008229.1AATC01008317.1 AATC01008371.1 AATC01008394.1 AATC01008454.1AATC01008467.1 AATC01008586.1 AATC01008590.1 AATC01008653.1AATC01008657.1 AATC01008704.1 AATC01008708.1 AATC01008769.1AATC01008790.1 AATC01008882.1 AATC01009143.1 AATC01009160.1AATC01009174.1 AATC01009216.1 AATC01009236.1 AATC01009264.1AATC01009285.1 AATC01009305.1 AATC01009318.1 AATC01009421.1AATC01009813.1 AATC01009852.1 AATC01010089.1 AATC01010227.1AATD01000362.1 AATD01000426.1 AATD01000446.1 AATD01000456.1AATD01000731.1 AATD01000753.1 AATD01000832.1 AATD01000900.1AATD01000922.1 AATD01001011.1 AATD01001014.1 AATD01001046.1AATD01001049.1 AATD01001073.1 AATD01001076.1 AATD01001178.1AATD01001275.1 AATD01001276.1 AATD01001302.1 AATD01001336.1AATD01001369.1 AATD01001378.1 AATD01001433.1 AATD01001449.1AATD01001466.1 AATD01001542.1 AATD01001607.1 AATD01001610.1AATD01001696.1 AATD01001850.1 AATD01001900.1 AATD01001975.1AATD01001999.1 AATD01002043.1 AATD01002092.1 AATD01002139.1AATD01002154.1 AATD01002285.1 AATD01002290.1 AATD01002310.1AATD01002409.1 AATD01002431.1 AATD01002452.1 AATD01002456.1AATD01002715.1 AATD01002795.1 AATD01002800.1 AATD01002847.1AATD01002906.1 AATD01002933.1 AATD01002996.1 AATD01003001.1AATD01003019.1 AATD01003055.1 AATD01003120.1 AATD01003121.1AATD01003190.1 AATD01003252.1 AATD01003323.1 AATD01003334.1AATD01003349.1 AATD01003352.1 AATD01003428.1 AATD01003448.1AATD01003470.1 AATD01003518.1 AATD01003541.1 AATD01003550.1AATD01003589.1 AATD01003623.1 AATD01003628.1 AATD01003709.1AATD01003727.1 AATD01003753.1 AATD01003868.1 AATD01003892.1AATD01003900.1 AATD01003917.1 AATD01004018.1 AATD01004028.1AATD01004055.1 AATD01004112.1 AATD01004145.1 AATD01004162.1AATD01004190.1 AATD01004222.1 AATD01004286.1 AATD01004364.1AATD01004455.1 AATD01004512.1 AATD01004556.1 AATD01004578.1AATD01004625.1 AATD01004716.1 AATD01004764.1 AATD01004770.1AATD01004772.1 AATD01004785.1 AATD01004896.1 AATD01004907.1AATD01004927.1 AATD01004971.1 AATD01004992.1 AATD01004997.1AATD01005014.1 AATD01005033.1 AATD01005042.1 AATD01005128.1AATD01005130.1 AATD01005136.1 AATD01005162.1 AATD01005189.1AATD01005195.1 AATD01005280.1 AATD01005285.1 AATD01005426.1AATD01005451.1 AATD01005477.1 AATD01005616.1 AATD01005687.1AATD01005696.1 AATD01005757.1 AATD01005790.1 AATD01005853.1AATD01005866.1 AATD01005911.1 AATD01005988.1 AATD01006038.1AATD01006057.1 AATD01006070.1 AATD01006104.1 AATD01006128.1AATD01006159.1 AATD01006192.1 AATD01006234.1 AATD01006242.1AATD01006291.1 AATD01006313.1 AATD01006366.1 AATD01006447.1AATD01006506.1 AATD01006516.1 AATD01006555.1 AATD01006575.1AATD01006576.1 AATD01006592.1 AATD01006630.1 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AATE01008403.1 AATE01008413.1AATE01008415.1 AATE01008506.1

The arrays may be utilized in several suitable applications. Forexample, the arrays may be used in methods for detecting associationbetween two or more biomolecules. This method typically comprisesincubating a sample with the array under conditions such that thebiomolecules comprising the sample may associate with the biomoleculesattached to the array. The association is then detected, using meanscommonly known in the art, such as fluorescence. “Association,” as usedin this context, may refer to hybridization, covalent binding, or ionicbinding. A skilled artisan will appreciate that conditions under whichassociation may occur will vary depending on the biomolecules, thesubstrate, and the detection method utilized. As such, suitableconditions may have to be optimized for each individual array created.

In yet another embodiment, the array may be used as a tool in a methodto determine whether a compound has efficacy for treatment of obesity oran obesity-related disorder in a host. Alternatively, the array may beused as a tool in a method to determine whether a compound increases ordecreases the relative abundance of Bacteriodes or Firmicutes in asubject. Typically, such methods comprise comparing a plurality ofbiomolecules of the host's microbiome before and after administration ofa compound, such that if the abundance of biomolecules associated withobesity decreased after treatment, or the abundance of biomoleculesindicative of Bacteroides increases, or the abundance of biomoleculesindicative of Firmicutes decreases, the compound may be efficacious intreating obesity in a host.

The array may also be used to quantitate the plurality of biomoleculesof the host microbiome before and after administration of a compound.The abundance of each biomolecule in the plurality may then be comparedto determine if there is a decrease in the abundance of biomoleculesassociated with obesity after treatment.

In some embodiments, the array may be used as a diagnostic or prognostictool to identify subjects that are susceptible to more efficient energyharvesting, and therefore, more susceptible to weight gain and/orobesity. Such a method may generally comprise incubating the array withbiomolecules derived from the subject's gut microbiome to determine therelative abundance of Bacteroidetes or Firmictues. In some embodiments,the array may be used to determine the relative abundance of Mollicutesin a subject's gut microbiome. Methods to collect, isolate, and/orpurify biomolecules from the gut microbiome of a subject to be used inthe above methods are known in the art, and are detailed in theexamples.

(b) Microbiome Profiles

The present invention also encompasses use of the microbiome as abiomarker to construct microbiome profiles. Generally speaking, amicrobiome profile is comprised of a plurality of values with each valuerepresenting the abundance of a microbiome biomolecule. The abundance ofa microbiome biomolecule may be determined, for instance, by sequencingthe nucleic acids of the microbiome as detailed in the examples. Thissequencing data may then be analyzed by known software, as detailed inthe examples, to determine the abundance of a microbiome biomolecule inthe analyzed sample. The abundance of a microbiome biomolecule may alsobe determined using an array described above. For instance, by detectingthe association between a biomolecules comprising a microbiome sampleand the biomolecules comprising the array, the abundance of a microbiomebiomolecule in the sample may be determined.

A profile may be digitally-encoded on a computer-readable medium. Theterm “computer-readable medium” as used herein refers to any medium thatparticipates in providing instructions to a processor for execution.Such a medium may take many forms, including but not limited tonon-volatile media, volatile media, and transmission media. Non-volatilemedia may include, for example, optical or magnetic disks. Volatilemedia may include dynamic memory. Transmission media may include coaxialcables, copper wire and fiber optics. Transmission media may also takethe form of acoustic, optical, or electromagnetic waves, such as thosegenerated during radio frequency (RF) and infrared (IR) datacommunications. Common forms of computer-readable media include, forexample, a floppy disk, a flexible disk, hard disk, magnetic tape, orother magnetic medium, a CD-ROM, CDRW, DVD, or other optical medium,punch cards, paper tape, optical mark sheets, or other physical mediumwith patterns of holes or other optically recognizable indicia, a RAM, aPROM, and EPROM, a FLASH-EPROM, or other memory chip or cartridge, acarrier wave, or other medium from which a computer can read.

A particular profile may be coupled with additional data about thatprofile on a computer readable medium. For instance, a profile may becoupled with data about what therapeutics, compounds, or drugs may beefficacious for that profile. Conversely, a profile may be coupled withdata about what therapeutics, compounds, or drugs may not be efficaciousfor that profile. Alternatively, a profile may be coupled with knownrisks associated with that profile. Non-limiting examples of the type ofrisks that might be coupled with a profile include disease or disorderrisks associated with a profile. The computer readable medium may alsocomprise a database of at least two distinct profiles.

Such a profile may be used, for instance, in a method of selecting acompound for treating obesity or an obesity-related disorder in a host.Generally speaking, such a method would comprise providing a microbiomeprofile from the host and providing a plurality of reference microbiomeprofiles, each associated with a compound, and selecting the referenceprofile most similar to the host microbiome profile, to thereby select acompound for treating obesity or an obesity-related disorder in thehost. The host profile and each reference profile may comprise aplurality of values, each value representing the abundance of amicrobiome biomolecule.

The microbiome profiles may be utilized in a variety of applications.For example, the microbiome profiles may be used in a method forpredicting risk for obesity or an obesity-related disorder in a host.The method comprises, in part, providing a microbiome profile from ahost, and providing a plurality of reference microbiome profiles, thenselecting the reference profile most similar to the host microbiomeprofile, such that if the host's microbiome is most similar to areference obese microbiome, the host is at risk for obesity or anobesity-related disorder. The microbiome profile from the host may bedetermined using an array of the invention. The reference profiles maybe stored on a computer-readable medium such that software known in theart and detailed in the examples may be used to compare the microbiomeprofile and the reference profiles.

The host microbiome may be derived from a subject that is a rodent, ahuman, a livestock animal, a companion animal, or a zoological animal.In one embodiment, the host microbiome is derived from a rodent, i.e. amouse, a rat, a guinea pig, etc. In another embodiment, the hostmicrobiome is derived from a human. In a yet another embodiment the hostmicrobiome is derived from a livestock animal. Non-limiting examples oflivestock animals include pigs, cows, horses, goats, sheep, llamas andalpacas. In still another embodiment, the host microbiome is derivedfrom a companion animal. Non-limiting examples of companion animalsinclude pets, such as dogs, cats, rabbits, and birds. In still yetanother embodiment, the host microbiome is derived from a zoologicalanimal. As used herein, a “zoological animal” refers to an animal thatmay be found in a zoo. Such animals may include non-human primates,large cats, wolves, and bears.

(c) Kits

The present invention also encompasses a kit for evaluating a compound,therapeutic, or drug. Typically, the kit comprises an array and acomputer-readable medium. The array may comprise a substrate, thesubstrate having disposed thereon at least one biomolecule that ismodulated in an obese host microbiome compared to a lean hostmicrobiome. The computer-readable medium may have a plurality ofdigitally-encoded profiles wherein each profile of the plurality has aplurality of values, each value representing the abundance of abiomolecule in a host microbiome detected by the array. The array may beused to determine a profile for a particular host under particularconditions, and then the computer-readable medium may be used todetermine if the profile is similar to known profile stored on thecomputer-readable medium. Non-limiting examples of possible knownprofiles include obese and lean profiles for several different hosts,for example, rodents, humans, livestock animals, companion animals, orzoological animals.

DEFINITIONS

The term “abundance” refers to the representation of a given phylum,order, family, or genera of microbe present in the gastrointestinaltract of a subject.

The term “activity of the microbiota population” refers to themicrobiome's ability to harvest energy.

The term “antagonist” refers to a molecule that inhibits or attenuatesthe biological activity of a Fiaf polypeptide and in particular, theability of Fiaf to inhibit LPL. Antagonists may include proteins such asantibodies, nucleic acids, carbohydrates, small molecules, or othercompounds or compositions that modulate the activity of a Fiafpolypeptide either by directly interacting with the polypeptide or byacting on components of the biological pathway in which Fiafparticipates.

The term “agonist” refers to a molecule that enhances or increases thebiological activity of a Fiaf polypeptide and in particular, the abilityof Fiaf to inhibit LPL. Agonists may include proteins, peptides, nucleicacids, carbohydrates, small molecules (e.g., such as metabolites), orother compounds or compositions that modulate the activity of a Fiafpolypeptide either by directly interacting with the polypeptide or byacting on components of the biological pathway in which Fiafparticipates.

The term “altering” as used in the phrase “altering the microbiotapopulation” is to be construed in its broadest interpretation to mean achange in the representation of microbes in the gastrointestinal tractof a subject. The change may be a decrease or an increase in thepresence of a particular microbial species, genus, family, order, orclass.

“BMI” as used herein is defined as a human subject's weight (inkilograms) divided by height (in meters) squared.

An “effective amount” is a therapeutically-effective amount that isintended to qualify the amount of agent that will achieve the goal of adecrease in body fat, or in promoting weight loss.

Fas stands for fatty acid synthase.

Fiaf stands for fasting-induced adipocyte factor.

LPL stands for lipoprotein lipase.

The term “obesity-related disorder” includes disorders resulting from,at least in part, obesity. Representative disorders include metabolicsyndrome, type II diabetes, hypertension, cardiovascular disease, andnonalcoholic fatty liver disease.

The term “metagenomics” refers to the application of modern genomicstechniques to the study of communities of microbial organisms directlyin their natural environments, by passing the need for isolation and labcultivation of individual species.

PPAR stands for peroxisome proliferator-activator receptor.

A “subject in need of treatment for obesity” generally will have atleast one of three criteria: (i) BMI over 30; (ii) 100 poundsoverweight; or (iii) 100% above an “ideal” body weight as determined bygenerally recognized weight charts.

As various changes could be made in the above compounds, products andmethods without departing from the scope of the invention, it isintended that all matter contained in the above description and in theexamples given below, shall be interpreted as illustrative and not in alimiting sense.

EXAMPLES

The following examples illustrate various iterations of the invention.

Example 1 Shotgun Sequencing of Microbiomes

To determine if microbial community gene content correlates with, and isa potential contributing factor to obesity, we characterized the distalgut microbiome of adult C57BL/6J mice homozygous for a mutation in theleptin gene (ob) that produces obesity, as well as the microbiomes oftheir lean (ob/+ and +/+) littermates by random shotgun sequencing oftheir cecal microbial DNA. Mice were used for these comparativemetagenomics studies to eliminate many of the confounding variables(environment, diet, and genotype) that would make such aproof-of-principle experiment more difficult to perform and interpret inhumans. The cecum was chosen as the gut habitat for sampling because itis an anatomically distinct structure, located between the distal smallintestine and colon that is colonized with sufficient quantities of areadily harvested microbiota for metagenomic analysis.

Animals.

All experiments involving mice were performed using protocols approvedby the Washington University Animal Studies Committee. Once C57BL/6Job/ob, ob/+, and +/+ littermates were weaned, they were housedindividually in microisolator cages where they were maintained in aspecified pathogen-free state, under a 12-h light cycle, and fed astandard polysaccharide-rich chow diet (PicoLab, Purina) ad libitum.Germ-free and colonized animals were maintained in gnotobioticisolators, under a strict 12-h light cycle and fed an autoclaved chowdiet (B&K Universal, East Yorkshire, U.K.) ad libitum. Fecal samples forbomb calorimetry were collected from mice at 8 or 14 weeks of age, afterwhich time animals were sacrificed.

Community DNA Preparation

The cecal contents used for community DNA sequencing and gaschromatography-mass spectrometry (GC-MS) were obtained, at eight weeksof age, from the same animals used for a previous PCR-based 16S rRNAsurvey of the gut microbiota (Ley et al. (2005) Proc. Natl. Acad, Sci.USA 102:11070-11075): samples had been stored at −80° C. (Table 1). Analiquot (˜10 mg) of each sample was suspended while frozen in a solutioncontaining 500 μL of extraction buffer [200 mM Tris (pH 8.0), 200 mMNaCl, 20 mM EDTA], 210 μL of 20% SDS, 500 μL of a mixture ofphenol:chloroform:isoamyl alcohol (25:24:1)], and 500 μL of a slurry of0.1-mm-diameter zirconia/silica beads (BioSpec Products, Bartlesville,Okla.). Microbial cells were then lysed by mechanical disruption with abead beater (BioSpec Products) set on high for 2 min (23° C.), followedby extraction with phenol:chloroform:isoamyl alcohol, and precipitationwith isopropanol. In order to perform pyrosequencing, DNA was purifiedfurther using the Qiaquick gel extraction kit (Qiagen).

Shotgun Sequencing and Assembly of Cecal Microbiomes

DNA samples were used to construct plasmid libraries for 3730x1capillary-based sequencing. Pyrosequencing was performed as previouslydescribed (Margulies at al. (2005) Nature 437:376-380). Briefly, sampleswere nebulized to 200 nucleotide fragments, ligated to adaptors, fixedto beads, suspended in a PCR reaction mixture-in-oil emulsion,amplified, and sequenced using a GS20 pyrosequencer (454 Life Sciences,Branford, Conn.). The Newbler de novo shotgun sequence assembler (454Life Sciences) was used to assemble sequences based on flowgram signalspace. This process included overlap generation, contig layout, andconsensus generation. The resulting GS20 contigs were then broken intolinked sequences to generate pseudo paired-end reads, and aligned with3730x1 reads using PCAP (Huang et al. (2003) Genome Res. 13:2164-2170).

Sequences were aligned to reference genomes using the PROmer script inMUMmer Kurtz et al. (2004) Genome Biol. 5:R12) (version 3.18). Capillarysequencer reads from each microbiome, the finished genome of the humangut-derived Bacteroides thetaiotaomicron type strain ATCC29148 (Xu etal. (2003) Science 299:2074-2076), and a deep draft genome of the humangut-derived Eubacterium rectale type strain ATCC33656(gordonlab.wustl.edu/supplemental/Turnbaugh/obob/) were used as areference for the pyrosequencer datasets. Coverage was calculated bydividing the sum of all alignment lengths by the length of the referencegenome.

Whole Genome Sequencing and Annotation

A draft assembly of Eubacterium rectale ATCC33656 was generated fromAB37301×1 paired end-reads of inserts in whole genome shotgun plasmidand fosmid libraries, as well as from reads produced by the GS20pyrosequencer. Sequences were assembled using Newbler and PCAP (seeabove) and ORFS predicted with Glimmer3.01 (Delcher et al. (1999) Nucl.Acids Res, 27:4636-4641) (maximum overlap of 100, minimum length of 110and a threshold of 30). Each predicted gene sequence was translated, andthe resulting protein sequence assigned to InterPro numbers usingInterProScan (Mulder et al. (2005) Nucl. Acids Res. 33:D201-205)(Release 12.0).

TABLE 1 Nomenclature used to designate metagenomic datasets obtainedfrom the cecal microbiota of C57BL/6J ob/ob, ob/+, and +/+ littermates.Figure Metagenome 16S rRNA survey Tree Host label label Litter label¹label¹ genotype ob1 PT6 1 C23 M2B-4 ob/ob ob2 PT4 2 C18 M1-2 ob/ob lean1PT3 1 C21 M2B-1 +/+ lean2 PT8 2 C15 M1-3 ob/+ lean3 PT2 2 C16 M1-4 +/+¹Samples from previous 16S rRNA survey: (Ley et al. (2005) Proc. Natl.Acad. Sci. USA 102: 11070-11075.

Results.

Bulk DNA was prepared from the cecal contents of two ob/ob and +/+littermate pairs. A lean ob/+ mouse from one of the litters was alsostudied. All cecal microbial community DNA samples were analyzed using a3730x1 capillary sequencer [10,500±431 unidirectional reads/dataset;752±13.8 (s.e.m.) nucleotides/read; 39.5 Mb from all five plasmidlibraries]. Material from one of the two obese and lean sibling pairswas also analyzed using a highly parallel 454 Life Sciences GS20pyrosequencer: three runs for the +/+ mouse (known as lean1), and tworuns for its ob/ob littermate (ob1) produced a total of 160 Mb ofsequence [345,000±23,500 unidirectional reads/run; 93.1±1.56nucleotides/read] (Tables 2 and 3). Both sequencing platforms haveunique advantages and limitations: capillary sequencing allows moreconfidant gene calling (FIG. 1) but is affected by cloning bias whilepyrosequencing can achieve higher sequence coverage with no cloningbias, but produces shorter reads (Table 2). The three pyrosequencer runsof the lean1 cecal microbiome (94.9 Mb) yielded 0.44× coverage (based onPROmer sequence alignments) of the 3730x1-derived sequences obtainedfrom the same sample (8.23 Mb), while the two pyrosequencer runs of themicrobiome of its ob/ob littermate (ob1, 65.4 Mb) produced 0.32×coverage of the corresponding 3730x1 sequences (8.19 Mb).

TABLE 2 Sequencing results for each cecal microbiome. Average NumberMicrobiome Sequencer read length of reads Sequence lean1 GS20 90.91,046,611 94,913,476 ob1 GS20 96.4 677,384 65,370,448 lean1 3730x1 76510,752 8,227,047 lean2 3730x1 782 11,136 8,705,876 lean3 3730x1 70610,752 7,590,528 ob1 3730x1 735 11,136 8,185,880 ob2 3730x1 771 8,8326,811,035 TOTAL 1,776,603 199,804,290

TABLE 3 Assembly of reads from capillary sequencer and pyrosequencerdatasets. N50 Average contig contig Contiged Largest length SampleSequencer Contigs length bases¹ Assembly (kb)² lean1 GS20 102,299 11711,966,580 2,793 0.109 ob1 GS20 56,425 116 6,518,469 2,174 0.109 lean13730x1 167 1527 254,985 5,500 1.62 lean2 3730x1 407 1598 650,499 5,5221.71 lean3 3730x1 224 1528 342,172 3,281 1.59 ob1 3730x1 320 1393445,814 3,225 1.49 ob2 3730x1 269 1644 442,210 4,186 1.70 All 3730x12,575 1734 4,465,685 11,213 1.78 All GS20 159,245 118 18,809,438 2,7080.110 All GS20 and 13,667 898 12,275,469 14,755 0.903 3730x1 ¹Contigedbases refers to the combined length of all contigs. ²N50 contig lengthrefers to the length of the contig, such that 50% of the total contigedbases are present in contigs of greater or equal size.

Example 2 Taxonomic Analysis of Microbiomes

Database Search Parameters

NCBI BLAST was used to query the nonredundant database (NR), theSTRING-extended COG database (179 microbial genomes, version 6.3) (vonMering et al. (2005) Nucl. Acids Res. 33:D433-437), a databaseconstructed from 334 genomes available through KEGG (version 37)(Kanehisa et al. (2004) Nucl. Acids Res 32: D277-280), and the RibosomalDatabase Project database (RDP, version 9.33) (Cole et al. (2005) Nucl.Acids Res33:D294-296). Reads with multiple COG/KO hits were counted oncefor each classification scheme. KO hits were also categorized into CAZyfamilies (afmb.cnrs-mrs.fr/CAZY/). KEGG pathway maps are availableon-line (gordonlab.wustl.edu/supplemental/Turnbaugh/obob/). NR, COG, andKEGG comparisons were performed using NCBI BLASTX. RDP comparisons wereperformed using NCBI BLASTN, and microbiomes were directly comparedusing TBLASTX. A cutoff of e-value <10⁻⁵ was used for EGT assignmentsand sequence comparisons DeLong et al. (2006) Science 311:496-503)(corresponds to a p-value cutoff of 10⁻¹² against the NR and KEGGdatabases, and 10⁻¹¹ against the COG database). Given this cutoff, wewould only expect three false EGT assignments in our combined analysesdue to random chance. We also re-analyzed the data using a morestringent cutoff (Tringe et al. (2005) Science 308:554-557) (e-value<10⁻⁸).

Taxonomic Assignments of Shotgun 16S rRNA Gene Fragments

Shotgun reads containing a 16S rRNA fragment were identified by BLASTXcomparison of each microbiome to the RDP database. 16S rRNA genefragments were then aligned using the NASTA multi-aligner (DeSantis etal. (2006) Nucl. Acids Res. 34:W394-399) with a minimum template lengthof 20 bases and a minimum percent identity of 75%. The resultingalignment was then imported into an ARB neighbor-joining tree andhypervariable regions were masked using the lanemaskPH filter (Ludwig etal. (2004) Nucl. Acids Res. 32:1363-1371). Direct BLAST taxonomicassignments were performed through BLASTX comparisons of each microbiomeand the NR database. Best-BLAST-hits with an e-value <10⁻⁵ were used toassign each read to a given species.

Estimating the Total Number of Orthologous Groups

The total estimated number of COGs and NOGs (Non-supervised OrthologousGroups) in each sample was calculated using the lower-limit of the Chao195% confidence interval in EstimateS (Version 7.5, R. K. Colwell,purl.ocic.org/estimates), based on the number of EGTs assigned to eachorthologous group. The number of missed groups was calculated bysubtracting the estimated total (Chao1 lower-limit) from the observednumber of groups.

Direct comparisons of microbiome sequences Microbiomes sequenced usingthe 3730x1 instrument were evaluated by reciprocal pairwise TBLASTXcomparisons (DeLong et al. (2006) Science 311:496-503). 8,832 reads wereused from each microbiome to limit artifacts that arise from differentsized datasets. Each possible pairwise comparison was made by using aBLAST database constructed from each microbiome. Samples were clusteredbased on the cumulative pairwise BLAST score. An estimate of distancewas constructed using the D2 normalization and genome conservationapproach previously used for genome clustering (Kunin et al. (2005)Nucl. Acids Res. 33:616-621) This method calculates a distance scorebased on the minimum cumulative BLAST score (sum of all best-BLAST-hitscores) between two microbiomes and the weighted average of bothself-self comparisons (D2=−ln(min S_(1v2), S_(2v1)/average). Theweighted average is calculated usingaverage=squareroot(2)*S_(1v1)*S_(2v2)/squareroot (S_(1v1) ²+S_(2v2) ²).The resulting distances were used to create a distance matrix. A treewas constructed using NEIGHBOR(PHYLIP version 3.64; kindly provided byJ. Felsenstein, Department of Genome Sciences, University of Washington,Seattle), and was viewed using Treeview X (Page (1996) Comput. Appl.Biosci. 12:357-358).

Results.

Environmental gene tags (EGTs) are defined as sequencer reads assignedto the NCBI non-redundant (NR), Clusters of Orthologous Groups (COG), orKyoto Encyclopedia of Genes and Genomes (KEGG) databases (FIG. 2A; FIG.3 Table 4). Averaging results from all datasets, 94% of the EGTsassigned to NR were bacterial, 3.6% were eukaryotic (0.29% Mus musculus;0.36% fungal), 1.5% were archaeal (1.4% Euryarcheota; 0.07%Crenarcheota), and 0.61% were viral (0.57% dsDNA viruses) (Table 5). Therelative abundance of the eight bacterial divisions identified from EGTsand 16S rRNA gene fragments was comparable to our previous PCR-derived,16S rRNA gene sequence-based surveys of these cecal samples, includingthe increased ratio of Firmicutes to Bacteroidetes in obese versus leanlittermates (FIG. 3). In addition, comparisons of the lean1 and ob1reads obtained with the pyrosequencer against the finished genome of B.thetaiotaomicron ATCC29148, and a deep draft genome assembly ofEubacterium rectale ATCC33656 (N50 contig size 75.9 kB;gordonlab.wustl.edu/supplemental/Turnbaugh/obob/) provided independentconfirmation of the greater relative abundance of Firmicutes in theob/ob microbiota. These organisms were selected for comparison becauseboth are prominently represented in the normal human distal gutmicrobiota (Eckburg et al. (2005) Science 308:1635-1638) while speciesrelated to B. thetaiotaomicron and E. rectale are members of the normalmouse distal gut microbiota (Gill et al. (2006) Science 312:1355-1359).The ratio of sequences homologous to the E. rectale versus B.thetaiotaomicron genome was 7.3 in the ob1 cecal microbiome compared to1.5 in the lean1 microbiome.

There were more EGTs that matched Archaea (Euryarchaeota andCrenarchaeota) in the cecal microbiome of ob/ob mice compared to theirlean ob/+ and +/+ littermates (binomial test of pooled obese versuspooled lean capillary sequencing derived microbiomes, P<0.001) (Table5). Methanogenic archaea increase the efficiency of bacterialfermentation by removing one of its end products, H₂. Our recent studiesof gnotobiotic normal mice colonized with the principal methanogenicarchaeon in the human gut, Methanobrevibacter smithii, and/or B.thetaiotaomicron revealed that co-colonization not only increases theefficiency, but also changes the specificity of bacterial polysaccharidefermentation, leading to a significant increase inadiposity comparedwith mice colonized with either organism alone (Samuel and Gordon (2006)Proc. Natl. Acad. Sci. USA 103:10011-10016).

TABLE 4 Number of EGTs assigned to the NR, COG, and/or KEGG databases.Total Total NR COG Total KO Total Percent Microbiome EGTs EGTs EGTs EGTsunassigned lean1 (GS20) 48,625 51,481 28,359 56,599 94.6 ob1 (GS20)33,360 32,819 18,308 39,058 94.2 lean1 (3730xl) 7,973 7,970 2,810 8,46221.3 lean2 (3730xl) 7,309 7,687 2,723 8,170 26.6 lean3 (3730xl) 7,0427,119 2,562 7,616 29.2 ob1 (3730xl) 7,331 7,299 2,639 7,859 29.4 ob2(3730xl) 6,008 6,016 2,053 6,425 27.3

TABLE 5 Percentage of total assigned reads among each taxonomic domainbased on BLASTX searches of the NR database with an e-value cutoff≦10⁻⁵. lean1 lean1 lean2 lean3 ob1 ob1 ob2 Domain 3730x1 GS20 3730x13730x1 3730x1 GS20 3730x1 Archaea  1.28  0.658  1.55  1.59  2.07  1.23 2.08 Bacteria 95.8 97.9 90.7 95.1 94.4 93.4 92.9 Eukaryota  2.36  1.39 7.36  2.74  2.77  4.15  4.19 (Viruses)  0.527  0.065  0.383  0.611 0.709  1.21  0.782

Example 3 Comparative Metagenomic Analysis

Clustering of Microbiomes Based on Predicted Metabolic function

Microbiomes were clustered based on the percent representation of EGTsassigned to each COG, KEGG pathway, and phylotype (genome in NR) usingCluster3.0. Percent representation was calculated as the number of EGTsassigned to a given group divided by the number of EGTs assigned to allgroups. Single linkage hiearchical clustering via Pearson's correlationwas performed on each dataset, and the results were visualized by usingthe Treeview Java applet (Saldanha (2004) Bioinformatics 20:3246-3248).Principal Component Analysis was also performed based on the percentrepresentation of EGTs assigned to KEGG pathways (Cluster3.0) (Dailey etal. (1987) J. Bacteriol. 169:917-919), and the data were graphedaccording to the first two coordinates.

Identification of Statistically Enriched and Depleted Metabolic groups

Two methods were used to determine statistically enriched or depletedmetabolic groups: the cumulative binomial distribution ((Gill et al.(2006) Science 312:1355-1359) and a bootstrap analysis (DeLong et al.(2006) Science 311:496-503; Rodriguez-Brito et al. (2006) BMCBioinformatics 7:162). The cumulative binomial distribution was used forpairwise comparisons of microbiome COG, KEGG, and taxonomic assignments.The calculation uses the following inputs: number of successes formicrobiome 1 (number of EGTs assigned to a given group), number oftrials for microbiome 1 (total number of EGTs assigned to all groups),and the expected frequency (number of successes/number of trials formicrobiome 2). The probability of having less than or equal to thenumber of observed EGTs in a given group was then calculated using thecumulative binomial distribution. Depletion was defined as having aprobability less than 0.05, 0.01, or 0.001 assuming p equals theexpected frequency and that the expected frequency is normallydistributed. Enrichment was defined as having a probability of greaterthan 0.95, 0.99, or 0.999 given the same assumptions. To minimize falsenegatives, no corrections for multiple sampling were made. To limitfalse positives resulting from low sampling, only groups with at leastone hit in each microbiome were evaluated.

Xipe (Rodriguez-Brito, version 0.2) (Rodriguez-Brito et al. (2006) BMCBioinformatics 7:162) was employed for bootstrap analyses of KEGGpathway enrichment and depletion, using the following parameters: 10,000samples, 10,000 repeats, and three confidence levels (95%, 99%, and99.9%). Briefly, a dataset composed of the number of EGTs assigned toeach KEGG pathway was sampled with replacement from each microbiome10,000 times. The difference between the number of EGTs per pathway inthe first microbiome, and the number of EGTs per pathway in the secondmicrobiome, was calculated for each group. This process was repeated10,000 times and the median difference calculated for each pathway. Aconfidence interval was determined by pooling both datasets andcomparing 10,000 random samples to 10,000 other random samples. Groupswith a larger median difference between microbiomes than the confidenceinterval were considered significantly different.

Biochemical Analyses

Short-chain fatty acids (SCFAs) were measured in nine cecal samples (4lean, 5 obese) obtained from nine mice that had been used for ourprevious 16S rRNA gene sequence-based survey [animals C1, C3, C4, C9,C10, C13, C15 (lean2), C17, and C22 (Ley et al. (2005) Proc. Natl. Acad.Sci. USA 102:11070-11075)]. Two aliquots of each sample were evaluated.SCFA levels were quantified according to previously published protocols(Samuel and Gordon (2006) Proc. Natl. Acad. Sci. USA 103:10011-10016):i.e., double diethyl ether extraction of deproteinized cecal contentsspiked with isotope-labeled internal SCFA standards; derivatization ofSCFAs with N-tert-butyldimethylsilyl-Nmethyltrifluoracetamide (MTBSTFA);and GC-MS analysis of the resulting TBDMS derivatives.

Bomb calorimetry was performed on 44 fecal samples collected from 22mice (9 lean, 13 obese). Each mouse was transferred to a clean cage for24 hours, at which point fecal samples were collected and oven dried at60° C. for 48 hours. Gross energy content was measured using a semimicrooxygen bomb calorimeter, calorimetric thermometer, and semimicro oxygenbomb (Models 6725, 6772 and 1109, respectively, from Parr InstrumentCo.). The calorimeter energy equivalent factor was determined usingbenzoic acid standards. The mean of each distribution was compared usinga two-tailed Student's t-Test (p<0.05).

Results.

Using reciprocal TBLASTX comparisons, we found that theFirmicutes-enriched microbiomes from ob/ob hosts clustered together(FIG. 4A). Likewise, Principal Component Analysis of EGT assignments toKEGG pathways revealed a correlation between host genotype and the genecontent of the microbiome (FIG. 4B). Reads were then assigned to COGsand KOs (KEGG orthology terms) by BLASTX comparisons against theSTRING-extended COG database, and the KEGG Genes database (version 37).We tallied the number of EGTs assigned to each COG or KEGG category, andused the cumulative binomialdistribution, and a bootstrap analysis, toidentify functional categories with significant differences in theirrepresentation in both sets of obese and lean littermates.

As noted above, capillary sequencing requires cloned DNA fragments: thepyrosequencer does not, but produces relatively short read lengths.These differences are a likely cause of the shift in relative abundanceof several COG categories obtained using the two sequencing methods forthe same sample (FIG. 2B). Nonetheless, comparisons of the cecalmicrobiomes of lean versus obese littermates sequenced with eithermethod revealed similar differences in their functional profiles (FIG.2C).

The ob/ob microbiome is enriched for EGTs encoding many enzymes involvedin the initial steps in breaking down otherwise indigestible dietarypolysaccharides, including KEGG pathways for starch/sucrose metabolism,galactose metabolism, and butanoate metabolism (FIGS. 2D, 5; Table 6).EGTs representing these enzymes were grouped according to theirfunctional classifications in the Carbohydrate Active Enzymes (CAZy)database (afmb.cnrsmrs. fr/CAZY/). The ob/ob microbiome is enriched(P<0.05) for eight glycoside hydrolase families capable of degradingdietary polysaccharides including starch (Families 2, 4, 27, 31, 35, 36,42 and 68 which contain alpha-glucosidases, alphagalactosidases, andbeta-galactosidases). Finished genome sequences of prominent human gutFirmicutes have not been reported. However, our analysis of the draftgenome of E. rectale has revealed 44 glycoside hydrolases, including asignificant enrichment for glycoside hydrolases involved in thedegradation of dietary starches [CAZy Families 13 and 77 which containalpha-amylase and amylomaltase; P<0.05 based on binomial test of E.rectale versus the finished genomes of Bacteroidetes (Bacteroidesthetaiotaomicron ATCC29148, B. fragilis NCTC9343, B. vulgatus ATCC8482and B. distasonis ATCC8503].

EGTs encoding proteins that import the products of these glycosidehydrolases (ABC transporters), metabolize them [e.g., alpha- andbeta-galactosidases (KO7406/7 and KO1190)], and generate the major endproducts of fermentation, butyrate and acetate [KEGG ‘Butanoatemetabolism’ pathway; pyruvate formate-lyase (KO0656); andformate-tetrahydrofolate ligase (KO1938; second enzyme in thehomoacetogenesis pathway for converting CO2 to acetate)], are alsosignificantly enriched in the ob/ob microbiome (binomial comparison ofpyrosequencer-derived ob1 and lean1 datasets, P<0.05) (FIGS. 2D, 5;Table 6).

As predicted from our comparative metagenomic analyses, the ob/ob cecumhas an increased concentration of the major fermentation end-productsbutyrate and acetate (FIG. 6A). This observation is also consistent withthe fact that many Firmicutes are butyrate producers. Moreover, bombcalorimetry revealed that ob/ob mice have significantly less energyremaining in their feces relative to their lean littermates (FIG. 6B).

TABLE 6 KEGG pathways enriched in the pooled ob/ob cecal microbiomerelative to the pooled lean cecal microbiome (capillary sequencingdatasets, ob1 + ob2 vs. lean1 + lean2 + lean3, binomial test, P < 0.05).KEGG Category KEGG Pathway¹ Carbohydrate Metabolism Starch and sucrosemetabolism Aminosugars metabolism Nucleotide sugars metabolism AminoAcid Metabolism Lysine biosynthesis Metabolism of Other Amino AcidsD-Alanine metabolism Glycan Biosynthesis and Metabolism N-Glycandegradation Glycosaminoglycan degradation Glycosphingolipid metabolismBiosynthesis of Polyketides and Polyketide sugar unit biosynthesisNonribosomal Peptides Biosynthesis of vancomycin group antibioticsTranscription Other and unclassified family transcriptional regulatorsFolding, Sorting and Degradation Type III secretion system MembraneTransport ABC transporters Phosphotransferase system (PTS) SignalTransduction Two-component system Cell Motility Bacterial chemotaxisFlagellar assembly Bacterial motility proteins Cell Growth and DeathSporulation ¹Only pathways with greater than ten hits in both pooleddatasets are shown.

Example 4 Microbiota Transplantation

Microbiota Transplantation Experiments

Germ-free C57BL/6J mice (8-9 weeks old) were colonized with a cecalmicrobiota obtained from either a lean (+/+) or an obese (ob/ob)C57BL/6J donor (n=1 donor and 4-5 recipients/treatment group/experiment;2 independent experiments). Recipient mice were anesthetized at 0 and 14days post colonization with an i.p. injection of ketamine (10 mg/kg bodyweight) and xylazine (10 mg/kg) and total body fat content was measuredby dual-energy x-ray absorptiometry (Lunar PIXImus Mouse, GE MedicalSystems) using previously described protocols (Bernal-Mizrachi et al(2002) Arterioscler. Thromb. Vasc. Biol. 22:961-968). Donor mice weresacrificed at day 0 and recipient mice after the final DEXA on day 14.

16S rRNA Sequence-Based Surveys of the Cecal Microbiotas ofConventionalized Mice

Cecal contents were recovered at the time of sacrifice by manualextrusion and frozen immediately at −80° C. DNA was prepared by beadbeating, phenol/chloroform extraction, and gel purification (see above).Five replicate PCRs were performed for each mouse. Each 25 μl reactioncontained 50-100 ng of purified DNA from cecal contents, 10 mM Tris (pH8.3), 50 mMKCl, 2 mM MgSO4, 0.16 μM dNTPs, 0.4 μM of thebacteria-specific primer 8F (5′-AGAGTTTGATCCTGGCTCAG-3′), 0.4 μM of theuniversal primer 1391 R (5′-GACGGGCGGTGWGTRCA-3′), 0.4 M betaine, and 3units of Taq polymerase (Invitrogen). Cycling conditions were 94° C. for2 min, followed by 35 cycles of 94° C. for 1 min, 55° C. for 45 sec, and72° C. for 2 min, with a final extension period of 20 min at 72° C.Replicate PCRs were pooled, concentrated with Millipore columns(Montage), gel-purified with the Qiaquick kit (Qiagen), cloned into TOPOTA pCR4.0 (Invitrogen), and transformed into E. coli TOP10 (Invitrogen).For each mouse, 384 colonies containing cloned amplicons were processedfor sequencing.

Plasmid inserts were sequenced bidirectionally using vector-specificprimers and the internal primer 907R (5′-CCGTCAATTCCTTTRAGTTT-3′). 16SrRNA gene sequences were edited and assembled into consensus sequencesusing the PHRED and PHRAP software packages within the Xplorseq program(Papineau et al. (2006) Appl. Environ. Microbiol. 71:4822-4832).Sequences that did not assemble were discarded and bases with PHREDquality scores <20 were trimmed. Sequences were checked for chimerasusing Bellerophon (Huber et al. (2004) Bioinformatics 20:2317-2319) andsequences with greater than 95% identity to both parents were removed(n=535; 13% of aligned sequences). The final dataset (n=4,157 sequences;for ARB alignment and tree seegordonlab.wustl.edu/supplemental/Turnbaugh/obob/; for sequencedesignations see Table 7) was aligned using the on-line version of theNAST multialigner (DeSantis et al. (2006) Nucl. Acid Res. 34:W394-399)(minimum alignment length=1250; percent identity >75), hypervariableregions were masked using the lanemaskPH filter provided with the ARBdatabase (Ludwig et al. (2004) Nucl. acid Res. 32: 1363-1372, and thealigned sequences were added to the ARB neighbor-joining tree (based onpairwise distances with the Olsen correction) with the parsimonyinsertion tool. A phylogenetic tree containing all 16S rRNA genesequences was exported from ARB and clustered using online UniFrac(Lozupone et al. (2006) BMC Bioiniformatics 7:371) without abundanceweighting.

Results.

Together, these data are consistent with an overall increase in theability of the ob/ob microbiota to harvest energy from the diet. Thisnotion was tested experimentally by performing microbiotatransplantation experiments. Adult germ-free C57BL/6J mice werecolonized (by gavage) with a microbiota harvested from the cecum ofobese (ob/ob) or lean (+/+) donors (1 donor and 4-5 germ-free recipientsper treatment group per experiment; two independent experiments). 16SrRNA gene sequence-based surveys confirmed that the ob/ob donormicrobiota had a greater relative abundance of Firmicutes compared tothe lean donor microbiota (FIG. 7; Table 7). Furthermore, the ob/obrecipient microbiota had a significantly higher relative abundance ofFirmicutes compared to the lean recipient microbiota (p<0.05, two-tailedStudent's t-Test). UniFrac analysis of 16S rRNA gene sequences obtainedfrom the recipients' cecal microbiotas revealed that they clusteraccording to the input donor community (FIG. 7): i.e., the initialcolonizing community structure did not exhibit marked changes by the endof the two-week experiment. There was no statistically significantdifference in (i) chow consumption over the 14 day period [55.4±2.5 g(ob/ob) versus 54.0±1.2 g (+/+); caloric density of chow, 3.7 kcal/g],(ii) initial body fat (2.7±0.2 g for both groups as measured by dualenergy x-ray absorptiometry; DEXA), or (iii) initial weight between therecipients of lean and obese microbiotas. Strikingly, mice colonizedwith an ob/ob microbiota exhibited a significantly greater percentageincrease in body fat over two weeks than mice colonized with a +/+microbiota [FIG. 6C; 47±8.3 vs. 27±3.6 percentage increase or 1.3±0.2vs. 0.86±0.1 g fat (DEXA): at 9.3 kcal/g fat, this corresponds to adifference of 4 kcal or 2% of total calories consumed].

TABLE 7 16S rRNA gene-sequence libraries from microbiota transplantexperiments Host 16S gene Label in FIG. S4 ARB label Genotype sequenceslean donor 1 lean2 +/+ 166 ob/ob donor 1 obob1 ob/ob 199 ob/ob donor 2obob2 ob/ob 229 lean recipient 1 SWPT11 +/+ 248 lean recipient 2 SWPT13+/+ 265 lean recipient 3 SWPT18 +/+ 247 lean recipient 4 SWPT19 +/+ 278lean recipient 5 SWPT20 +/+ 271 ob/ob recipient 1 SWPT1 +/+ 219 ob/obrecipient 2 SWPT2 +/+ 268 ob/ob recipient 3 SWPT3 +/+ 280 ob/obrecipient 4 SWPT4 +/+ 272 ob/ob recipient 5 SWPT5 +/+ 290 ob/obrecipient 6 SWPT12 +/+ 197 ob/ob recipient 7 SWPT14 +/+ 272 ob/obrecipient 8 SWPT15 +/+ 198 ob/ob recipient 9 SWPT16 +/+ 258 TOTAL — —4,157

TABLE 8 KEGG pathways depleted in the pooled ob/ob cecal microbiomerelative to the pooled lean cecal microbiome (capillary sequencingdatasets, ob1 + ob2 vs. lean1 + lean2 + lean3, binomial test, P < 0.05).KEGG Category KEGG Pathway¹ Carbohydrate MetabolismGlycolysis/Gluconeogenesis Citrate cycle (TCA cycle) Pentose phosphatepathway Pentose and glucuronate interconversions Fructose and mannosemetabolism Energy Metabolism Carbon fixation Reductive carboxylate cycle(CO2 fixation) Pyruvate/Oxoglutarate oxidoreductases Lipid MetabolismFatty acid metabolism Nucleotide Metabolism Pyrimidine metabolism AminoAcid Metabolism Glutamate metabolism Glycine, serine and threoninemetabolism Cysteine metabolism Arginine and proline metabolismPhenylalanine, tyrosine and tryptophan biosynthesis Glycan BiosynthesisLipopolysaccharide biosynthesis and Metabolism Metabolism of CofactorsRiboflavin metabolism and Vitamins Folate biosynthesis TranslationRibosome Folding, Sorting and Other ion-coupled transporters Degradation¹Only pathways with greater than ten hits in both pooled datasets areshown.

TABLE 9 COG categories involved in information storage and cellularprocesses that are enriched or depleted in the pooled ob/ob cecalmicrobiome relative to the pooled lean cecal microbiome (capillarysequencing datasets, ob1 + ob2 vs. lean1 + lean2 + lean3, binomial test,P < 0.05). ENRICHED [K] Transcription [L] Replication, recombination,repair [Y] Nuclear structure [T] Signal transduction [M] Cellwall/membrane/envelope biogenesis [N] Cell motility DEPLETED [J]Translation [V] Defense mechanisms [O] Posttranslational modification,protein turnover, chaperones

Example 5 Human Gut Microbes Linked to Obesity

Sequence generation and analysis. All subjects gave written informedconsent before participating in this study, which was approved by theWashington University Human Studies Committee. We studied 12 men andwomen (21 to 65 years-old; body mass index (BMI) 30 to 43 kg/m2) whowere randomly assigned to one of two low calorie diets: either a fatrestricted (FAT-R; ˜30% of calories from fat) or acarbohydrate-restricted (CARB-R; ˜25% of calories from carbohydrates).The recommended caloric intake for women on either diet was 1200-1500kcal/d, and 1500-1800 kcal/d for men. The total fiber content of bothdiets was similar (˜10-15 g/day). A morning stool sample was collectedbefore and at 12, 26 and 52 weeks after starting diet therapy. Stool wasalso collected at 0 and 52 weeks from two healthy men (aged 32 and 36;BMI 23 kg/m2). DNA was extracted from morning stool specimens, andbacterial 16S rRNA gene sequences were generated with bacterial primersusing protocols described in Ley et al. (2005) v102, pg. 11070-75, withthe following modifications: (i) replicate PCR reaction mixtures werepooled, concentrated, purified using a Montage PCR cleanup kit(Millipore), and further purified (1% agarose gel electrophoresis) priorto cloning; (ii) three sequence reads were generated per cloned 16S rRNAgene amplicon using vector-specific primers and the internal primer 907R(see Ley et al., 2005 PNAS). 16S rRNA gene sequences were edited andassembled as outlined in Gell et al. Sequences were aligned using thenast online alignment tool (greengenes.lbl.gov/cgi-bin/nph-index.cgi),and checked for chimeras using Bellerophon (Huber (2004) Bioinformatics20:2317-2319). Non-chimeric sequences >800 bp (n=18, 348) were added toan existing Arb alignment using the parsimony insertion tool (Ludwig(2004) Nucleic Acids Res 32:1363-71). Distance matrices, with Olsencorrection, were generated in Arb. DOTUR was used (i) to clustersequences >1 kb (n=16,177) into OTUs by % pair-wise identity (% ID,using a furthest-neighbor algorithm and a precision of 0.01), and (ii)to generate Shannon's diversity index (Schloss (2005) Appl. Env. Micro.71:1501-6). We used UniFrac (Lozupone (2005) Appl. Env. Micro71:8228-35) to cluster the samples based on an Arb-generatedneighbor-joining tree. The alignment of the 18,348-sequence dataset isavailable at gordonlab.wustl.edu/microbial_ecology_human_obesity.Sequences have been deposited in GenBank under accession numbersDQ793220-DQ802819, DQ803048, DQ803139-DQ810181, DQ823640-825343.

Statistical Analyses.

Analysis of variance was conducted using a model comparison approach.The p-value associated with the correlation coefficient describing therelationship between the change in Bacteroidetes and the change inweight was generated by permutation analysis: values were scrambledrandomly and a R2 generated 10,000 times; the distribution of R2 valueswas used to assess the probability of obtaining the observed R2.

Results.

To explore the relationship between gut microbial ecology and body fatin humans, we studied 12 obese subjects randomly assigned to either afat-restricted (FAT-R) or carbohydrate-restricted (CARB-R) low caloriediet. The composition of their gut microbiotas was monitored over oneyear by sequencing 16S rRNA genes from stool samples.

Using ≧97% sequence identity in 16S rRNA gene sequence among individualsas a definition of a species, the resulting dataset of 18,348 bacterial16S rRNA sequences (Table 10) revealed that most (70%) of the 4,074identified species-level phylogenetic types (phylotypes) were unique toindividual subjects. Despite the marked interpersonal differences inspecies-level diversity, members of the Bacteroidetes and Firmicutesdivisions dominated the microbiota (92.6% of all 16S rRNA sequences).

Bacterial lineages were remarkably constant within subjects over time:communities from the same subject were generally more similar to oneanother than to communities from other subjects (FIG. 10A). Before diettherapy, obese subjects had fewer Bacteroidetes (p<0.001) and moreFirmicutes (p=0.002) than lean controls (FIG. 10B). Over time, therelative abundance of Bacteroidetes increased (% Bacteroidetes vs.weeks, p<0.001) and the abundance of Firmicutes decreased (% Firmicutesvs. weeks, p=0.002), irrespective of the type of diet (FIG. 10B).Remarkably, this change was division-wide, and not due to blooms orextinctions of specific bacterial species. Correspondingly, diversitylevels were constant over time. The increased Bacteroidetes abundancecorrelated directly with percent weight loss (R2=0.8 and 0.5 for theCARB-R and FAT-R diets, respectively; p<0.05; FIG. 10C), and not withchanges in calorie content over time (R²=0.06 and 0.09 for the CARB-Rand FAT-R diets, respectively). The correlation between Bacteroidetesabundance and weight loss was observed only after a threshold weightloss of 6% for FAT-R and 2% for CARB-R was attained.

Obesity is the only disease process that we are aware of where apronounced, division-wide change in microbial ecology has beenassociated with host pathology. As such, it represents an attractivemodel for studying the role of the microbiota in health and disease. Thefactors that drive shifts in representation at such broad taxonomiclevels must operate on highly conserved bacterial traits since they areshared by a great variety of phylotypes within the divisions. The guthabitat itself selects for specific ratios of divisions: microbiotastransplanted from a donor species to germ-free recipients of a differentspecies reconfigure to match the community structure normally occurringin the recipient. The coexistence of Bacteroidetes and Firmicutes in thegut implies minimized competition for resources by cooperation orspecialization: the obese gut possesses yet uncharacterized physical orchemical properties that tip the balance towards the Firmicutes.

The direct correlation between the abundance of the Bacteroidetes andthe amount of weight loss in obese subjects reveals a dynamic linkagebetween adiposity and gut microbial ecology. These findings, togetherwith results obtained from mice, suggest that intentional manipulationof gut microbial communities could be a new approach for treatingobesity.

TABLE 10 Sequence prefixes by library, and the number of sequences perlibrary 0 12 26 52 weeks weeks weeks weeks (N) Library Library LibraryLibrary Subject Sex Age Diet Group prefix N prefix N prefix N prefix N 1 F 57 FAT-R RL178 541 RL240 327 RL197 202 RL310 328  2 F 53 FAT-RRL182 178 RL242 296 RL205 277 RL305 346  3 F 54 FAT-R RL187 803 RL251287 RL200 335 RL385 274  4 F 48 FAT-R RL188 579 RL241 287 RL201 310RL311 244  5 M 55 FAT-R RL180 855 RL244 312 RL198 189 RL307 309  6 M 55FAT-R RL184 877 RL243 306 RL239 289 RL308 235  7 F 42 CARB-R RL176 543RL246 236 RL199 309  8 F 30 CARB-R RL179 767 RL245 215 RL202 271 RL386294  9 F 42 CARB-R RL181 539 RL248 302 RL206 325 RL302 337 10 F 49CARB-R RL183 481 RL247 309 RL303 254 11 F 35 CARB-R RL186 865 RL249 227RL203 304 RL306 331 12 M 54 CARB-R RL185 831 RL250 284 RL204 290 RL304300 13 M 32 CONTROL RL116 100 RL387 252 14 M 36 CONTROL RL117 93 RL388303 TOTAL 18,348

Example 6 Diet-Induced Obesity Alters Gut Microbial Ecology

The following materials and methods are also applicable to examples 7and 8.

Animals

All experiments involving mice were performed using protocols approvedby the Washington University Animal Studies Committee.

Conventionalization

Germ-free male 8-9 week old C57BL/6J mice were maintained in plasticgnotobiotic isolators, under a strict 12-h light cycle and fed anautoclaved low-fat, polysaccharide-rich chow diet (CHO) ad libitum[1,28]. Conventionalization was performed by harvesting cecal contentsfrom conventionally-raised animals, and introducing them, by gavage,into germ-free recipients, as described in ref. 4.

Conventionally-Raised Mice

Once C57BL/6J littermates were weaned, they were housed individually inmicroisolator cages where they were maintained in a specifiedpathogen-free state, under a 12-h light cycle, and fed a CHO diet(PicoLab, Purina), a high-fat/high-sugar Western diet (Harlan-TekladTD96132), a fat-restricted (FAT-R) diet (Harlan-Teklad TD05633), or acarbohydrate-restricted (CARB-R) diet (Harlan-Teklad TD05634) adlibitum.

Microbiota Transplantation Experiments

Adult germ-free C57BL/6J mice 8 weeks old were colonized with a cecalmicrobiota obtained from wild-type (+/+) C57BL/6J donor mice fed CHO,Western, FAT-R, or CARB-R diets. Recipient mice, maintained on a CHOdiet, were anesthetized at 0.5 and 14 days post colonization with anintraperitoneal injection of ketamine (10 mg/kg body weight) andxylazine (10 mg/kg) and total body fat content was measured bydual-energy x-ray absorptiometry (DEXA; Lunar PIXImus Mouse, GE MedicalSystems) [29]. Recipient mice were housed individually in microisolatorcages within gnotobiotic isolators throughout the experiment to avoidexposure to the microbiota of the other mice, and to allow the directmonitoring of the chow consumed by each mouse. Animals were sacrificedimmediately after the final DEXA on day 14.

Shotgun Sequencing and Assembly of Cecal Microbiomes

DNA samples were used to construct pOTw13-based libraries (GC10 cells,Gene Choice) for capillary-based sequencing with an ABI 3730x1instrument. Unidirectional (forward) sequencing reads were generatedfrom each library (an average of 10,600 reads/library). Reverse readswere also generated to improve assembly (768-1536 per library; total of7,680 reads;). Sequences were trimmed based on quality score and vectorsequences were removed prior to analysis (Applied Biosystems; KBBasecaller). Each dataset was assembled individually, in addition to acombined assembly of all seven datasets, using ARACHNE (parameters:maxcliq1=500; maxcliq2=500; genome size=1 Gb) [24]. ARACHNE was chosenbecause it has been shown to generate reliable contigs from complexsimulated metagenomic datasets [30]. Genes were predicted fromindividual sequencing reads and contigs using MetaGene [25].

Microbiome Functional Analysis

NCBI BLAST was used to query the STRING-extended COG database [19] andthe KEGG database (version 40) [20]. COG and KEGG comparisons wereperformed by using NCBI BLASTX employing default parameters. A cutoff ofe-value <10⁻⁵ was used for environmental gene tag (EGT) assignments andsequence comparisons. Predicted proteins were searched for conserveddomains and assigned functional identifiers with InterProScan (version4.3) [31]. Predicted glycoside hydrolases were confirmed based oncriteria used for the Carbohydrate Active Enzymes (CAZy) database(www.cazy.org/; Bernard Henrissat, personal communication).

Statistical Methods

χ² tests were performed on the number of gene assignments to a givenKEGG or STRING orthologous group in each microbiome relative to thenumber of gene assignments to all other groups. Xipe (version 2.4) [32]was employed for bootstrap analyses of KEGG pathway enrichment anddepletion, as described previously [2], using the parameters samplesize=10,000 and confidence level=0.90. ANOVA was performed using a modelcomparison approach [33], implemented with the linear regressionfunction in Excel (version 11.0, Microsoft). Student's t-tests wereutilized to identify statistically significant differences between twogroups. Data are represented as mean±SEM unless otherwise indicated. Thep-value associated with a given correlation coefficient (R²) wasgenerated by a permutation analysis, as described previously [9].Briefly, the values were scrambled randomly and an R² generated 10,000times; the resulting distribution of R² valueswas used to assess theprobability of obtaining the observed R².

Preparation of DNA from the Cecal Microbiota

Cecal contents were frozen at −80° C. immediately after sacrifice. Analiquot (˜10 mg) of each sample was then suspended, while frozen, in asolution containing 500 μl of extraction buffer [200 mM Tris (pH 8.0),200 mM NaCl, 20 mM EDTA], 210 μl of 20% SDS, 500 μl of a mixture ofphenol:chloroform:isoamyl alcohol (pH 7.9, 25:24:1), and 500 μl of aslurry of 0.1 mm-diameter zirconia/silica beads (BioSpec Products,Bartlesville, Okla.). Microbial cells were subsequently lysed bymechanical disruption with a bead beater (BioSpec Products) set on highfor 2 min at RT, followed by extraction with phenol:chloroform:isoamylalcohol (pH 7.9, 25:24:1), and precipitation with isopropanol. DNAobtained from ten separate 10 mg frozen aliquots of each cecal samplewere pooled (200 pg DNA) and used to construct plasmid libraries(pOTw13) for 3730x1 capillary-based metagenomic sequencing (see below).

16S rRNA Sequence-Based Surveys of the Distal Gut (Cecal) MouseMicrobiota

Five replicate PCR reactions were performed for each cecal DNA sample.Each 25 μl reaction contained 50-100 ng of purified DNA, 10 mM Tris (pH8.3), 50 mM KCl, 2 mM MgSO₄, 0.16 μM dNTPs, 0.4 μM of thebacteria-specific primer 8F (5′-AGAGTTTGATCCTGGCTCAG-3′), 0.4 μM of theuniversal primer 1391 R (5′-GACGGGCGGTGWGTRCA-3′), 0.4 M betaine, and 3units of Taq polymerase (Invitrogen). Cycling conditions were 94° C. for2 min, followed by 35 cycles of 94° C. for 1 min, 55° C. for 45 sec, and72° C. for 2 min, with a final extension period of 20 min at 72° C.Replicate PCRs were pooled and concentrated (Millipore; Montage PCRfilter columns). Full-length 16S rRNA gene amplicons (1.3 kb) were thengel-purified using the Qiaquick kit (Qiagen), subcloned into TOPO TApCR4.0 (Invitrogen), and the ligated DNA transformed into E. coli TOP10(Invitrogen). For each mouse, 384 colonies containing cloned ampliconswere processed for sequencing. Plasmid inserts were sequencedbi-directionally using vector-specific primers plus the internal primer907R (5′-CCGTCAATTCCTTTRAGTTT-3′).

16S rRNA gene sequences were edited and assembled into consensussequences using the PHRED and PHRAP software packages within theXplorseq program [39]. Sequences that did not assemble were discardedand bases with PHRED quality scores <20 were trimmed. Sequences werechecked for chimeras using Bellerophon version 2 [40] and sequences withgreater than 95% identity to both parents were removed (n=535; 13% ofaligned sequences). The final dataset (n=8,511 16S rRNA gene sequences;for sequence designations see Table 13) was aligned using the on-lineversion of the NAST multi-aligner [41] [minimum alignment length=1250nucleotides (500 for Rag1−/− data); percent identity >75]. Hypervariableregions were masked using the lanemaskPH filter provided within the ARBdatabase [42], and the aligned sequences added to the ARBneighbor-joining tree (based on pairwise distances with the Olsencorrection), using the parsimony insertion tool. A phylogenetic treecontaining all 16S rRNA gene sequences was then exported from ARB,clustered using online UniFrac [12] without abundance weighting, andvisualized with TreeView [43]. A distance matrix of all 16S rRNA genesequences was imported into DOTUR [13] for phylotype binning andmeasurements of diversity (e.g., the Shannon index).

Taxonomic Assignment of Shotgun Sequencing Reads

Quality-trimmed reads were assigned to reference genomes by comparisonwith the NCBI non-redundant database (NR version Apr. 19, 2007; BLASTXe-value <10⁻⁵; BLASTX parameters ‘−F F’). Sequences were assigned to thetaxonomic group (division, class, genus, etc.) that would include allsignificant hits using MEGAN (under the default parameters, only readswith a BLAST score 0% of the top score were included) [18]. Readscontaining a 16S rRNA fragment were identified by BLASTN comparison ofeach microbiome to the RDP database (version 9.33) [34]. 16S rRNA genefragments were then aligned using the NASTA multi-aligner [41] with aminimum template length of 400 bases and a minimum percent identity of75%. The resulting alignment was then imported into an ARBneighbor-joining tree and hypervariable regions masked using thelanemaskPH filter [42].

Transcriptional Profiling

A 10 mg aliquot of frozen cecal contents from a mouse fed the Westerndiet (sample ‘Western 3’) was immersed in 1 ml of RNAProtect (Qiagen),vortexed, centrifuged for 10 min at 5000×g, and the supernatant wasremoved. Microbial cells in the pellet were subsequently lysed bymechanical disruption with a bead beater (BioSpec Products) set on highfor 2 min at RT in a solution containing 500 μl of extraction buffer.RNA was extracted with phenol:chloroform:isoamyl alcohol (pH 4.5,125:24:1), precipitated with isopropanol, and further purified with (i)the RNeasy Mini Kit (Qiagen), (ii) on-column digestion with DNAsel(Qiagen), (iii) an additional DNAse treatment (DNAfree kit, Ambion), and(iv) passage through a RNeasy column (Qiagen).

A modification of the protocol included with the MessageAmpII-bacteriaKit (Ambion) that was developed at MIT [44], was used for mRNA-enrichedcDNA synthesis. cDNA was purified (Qiaquick, Qiagen) and subcloned intopSMART (10G Supreme Cells, Lucigen). Plasmid inserts from 384 randomlypicked colonies were sequenced (single unidirectional reads) usingvector specific primers and an ABI 3730x1 instrument. Sequences weretrimmed based on quality score and to remove vector sequences (AppliedBiosystems; KB Basecaller), and to remove poly(A) tails. Only sequenceswith a final length 80 bases were analyzed (average of 430 nucleotides).Sequences were annotated based on BLASTX (see above) and BLASTNcomparisons against the NCBI nucleotide database (version Sep. 26, 2007;BLASTN parameters ‘−F F’). 16S rRNA sequences were annotated based ontheir best-BLAST hit to 16S rRNA genes of known taxonomic origin(e-value <10⁻²⁵). Although rRNA gene fragments were the dominantsequence (90.6% of the high-quality reads), the library had a lowerabundance of rRNA transcripts than comparable libraries created directlyfrom total distal gut community RNA (99%; P. J. Turnbaugh and J. I.Gordon, unpublished data).

Results

The leptin deficient, ob/ob mouse model of obesity established acorrelation between host adiposity, microbial community structure, andthe efficiency of energy extraction from a standard, low-fat rodent chowdiet that was rich in plant polysaccharides, but it did not allow us toinvestigate the effects of manipulating diet, or diminishing hostadiposity on the gut microbiota and its microbiome. Furthermore, leptindeficiency is extremely rare in humans and is associated with a varietyof other host phenotypes [11]. Therefore, the following examples, turnsto a mouse model of diet-induced obesity (DIO) produced by consumptionof a prototypic high-fat/high-sugar Western diet, where all animals weregenetically identical, ‘inherited’ a similar microbiota, and where oncean obese state was achieved, specified diets could be imposed to reduceadiposity.

Ten germ-free male C57BL/6J mice were weaned onto a low-fat chow dietrich in structurally complex plant polysaccharides (‘CHO’ diet), andthen gavaged at 12 weeks of age with a distal gut (cecal) microbiotaharvested from a conventionally-raised donor (see Table 11 for thepercentage of calories derived from protein, carbohydrate, and fat).This process of ‘conventionalization’ was designed to insure that allrecipients inherited a similar microbiota. All recipients weresubsequently maintained in gnotobiotic isolators. Four weeks later, fiveof the conventionalized mice were switched to ‘Western’ diet high insaturated and unsaturated fats (41% of total calories) and the types ofcarbohydrates commonly used as human food additives [sucrose (18% ofchow weight), maltodextrin (12%), plus corn starch (16%); Tables 11 and12]. The remaining five mice were continued on the CHO diet. All micewere sacrificed eight weeks later (24 weeks after birth) (FIG. 11A).Mice on the Western diet gained significantly more weight than micemaintained on the CHO diet (5.3±0.8 g versus 1.5±0.2 g; p<0.05,Student's t-test) and had significantly more epididymal fat (3.7±0.5%versus 1.7±0.1% of total body weight; p<0.01, Student's t-test).

TABLE 11 Protein, carbohydrate, and fat composition of various mousechow diets Diet Protein^(a) CHO^(a) Fat^(a) kcal/g CHO^(b) 23.2 60.716.1 3.74 Western 18.7 40.7 40.7 4.49 FAT-R 18.7 60.0 21.3 3.95 CARB-R48.3 11.2 40.5 4.31 ^(a)values represent percentage of total kcal;^(b)B&K Universal autoclavable chow diet (Sonnenburg et al., (2006) PloSBiol 4: e413)

TABLE 12 Percent weight of chow ingredients Ingredient Western FAT-RCARB-R Casein 23.6 20.8 59.9 DL-Methionine 0.354 0.354 0.000 Sucrose,Cane 18.3 32.0 0.000 Corn Starch 16.0 16.0 0.000 Maltodextrin (Lo-Dex)12.0 12.0 11.0 Vegetable Oil 10.0 5.00 10.0 Beef Tallow 10.0 4.10 8.80Cellulose (Fiber) 4.00 4.00 4.00 Mineral Mix (AIN-93G-MX) 4.13 4.13 4.13Calcium Phosphate Dibasic 0.472 0.472 0.472 Vitamin Mix (Teklad 40060)1.18 1.18 1.18 Ethoxyquin (Antioxidant) 0.002 0.002 0.002 CalciumCarbonate 0.000 0.000 0.500

Cecal microbial community structure was defined in each mouse in each ofthe two groups by sequencing full-length 16S rRNA gene ampliconsproduced by PCR of community DNA (see Materials and Methods inSupporting Information; n=96-343 16S rRNA gene sequences defined permouse; Table 13). Communities were then compared using the UniFracmetric [12]. The premise of UniFrac is that two microbial communitieswith a shared evolutionary history will share branches on a 16S rRNAphylogenetic tree, and that the fraction of branch length shared can bequantified and interpreted as the degree of community similarity.

TABLE 13 16S rRNA gene-sequence libraries 16S gene Figure label ARBlabel Host Host diet sequences CARB 1 WD1 CONV-D wt CHO 96 CARB 2 WD2CONV-D wt CHO 343 CARB 3 WD3 CONV-D wt CHO 267 CARB 4 WD4 CONV-D wt CHO207 CARB 5 WD5 CONV-D wt CHO 216 Western 1 WD6 CONV-D wt Western 222Western 2 WD7 CONV-D wt Western 256 Western 3 WD8 CONV-D wt Western 221Western 4 WD9 CONV-D wt Western 220 Western 5 WD10 CONV-D wt Western 221Donor 1 WD11 CONV-R wt — 194 CARB-R 1 MD4 CONV-R wt DIO, CARB-R 185family 2 CARB-R 2 MD8 CONV-R wt DIO, CARB-R 233 family 1 CARB-R 3 MD9CONV-R wt DIO, CARB-R 184 family 1 CARB-R 4 MD21 CONV-R wt DIO, CARB-R259 family 2 CARB-R 5 MD23 CONV-R wt DIO, CARB-R 516 family 1 CARB-R 6MD26 CONV-R wt DIO, CARB-R 138 family 2 FAT-R 1 MD18 CONV-R wt DIO,FAT-R 241 family 2 FAT-R 2 MD19 CONV-R wt DIO, FAT-R 203 family 2 FAT-R3 MD24 CONV-R wt DIO, FAT-R 177 family 2 FAT-R 4 MD25 CONV-R wt DIO,FAT-R 162 family 2 FAT-R 5 MD27 CONV-R wt DIO, FAT-R 127 family 2Western 6 MD2 CONV-R wt DIO, Western 263 family 2 Western 7 MD6 CONV-Rwt DIO, Western 126 family 2 Western 8 MD7 CONV-R wt DIO, Western 176family 2 Western 9 MD20 CONV-R wt DIO, Western 233 family 2 Western 10MD22 CONV-R wt DIO, Western 193 family 2 CARB 6 myd1 CONV-R MyD88 −/−CHO 241 CARB 7 myd2 CONV-R MyD88 −/− CHO 260 CARB 8 myd3 CONV-R MyD88−/− CHO 266 Western 11 myd4 CONV-R MyD88 −/− Western 223 Western 12 myd5CONV-R MyD88 −/− Western 231 — rag2 CONV-R Rag1 −/− CHO 66 — rag3 CONV-RRag1 −/− CHO 103 — rag4 CONV-R Rag1 −/− Western 84 — rag5 CONV-R Rag1−/− Western 111 rag6 CONV-R Rag1 −/− Western 94 — CRWD2 CONV-R wtWestern 272 — CRWD4 CONV-R wt CHO 265 — CRWD5 CONV-R wt CHO 167 — CRWD6CONV-R wt Western 225

The results of UniFrac analysis revealed that the five Westerndiet-associated cecal communities were more similar to each other thanto the five lean gut communities (FIG. 12). As in the ob/ob model ofobesity, the Western diet-associated cecal community had a significantlyhigher relative abundance of the Firmicutes and a significantly lowerrelative abundance of the Bacteroidetes (FIG. 13A). Unlike the ob/obmicrobiota, the observed shift in the Firmicutes was not division-wide:the overall diversity of the Western diet microbiota droppeddramatically, due to a bloom in a single class of the Firmicutes—theMollicutes (FIG. 13B,C and 12). Using 99% sequence identity among 16SrRNA genes as a threshold cutoff, we identified 132 ‘strain’-levelphylotypes represented within the Mollicute bloom: the bloom wasdominated by six phylotypes that together comprised 81% of the Mollicutesequences (FIG. 14) [13]. Other Mollicutes phylogenetically related tothis Glade have been cultured from the human gut (e.g. Eubacteriumdolichum, E. cylindroides, and E. biforme) and observed in 16S rRNAdatasets generated from the fecal microbiota of obese humans [9].However, there are no reported cultured representatives of the dominantphylotypes observed in the DIO mouse model (FIG. 14).

To determine whether these diet-induced changes in gut microbial ecologyalso occur in mice exposed to microbes starting at birth, we conducted afollow-up study using a different experimental design. In this case,conventionally-raised C57BL/6J mice were weaned onto a Western or a CHOdiet and then maintained, in separate cages, on those diets for 8-9weeks (n=8 animals/group). All animals were sacrificed after 12 weeks ofage (FIG. 11B). Those on the Western diet gained significantly moreweight (13.8±0.9 g versus 10.9±0.9 g; p<0.05, Student's t-test) and hadsignificantly greater adiposity (epididymal fat pad weight was 3.0±0.2%of total body weight in the Western diet group versus 1.6±0.1% in theCHO group; p<0.001, Student's t-test). The cecal microbiota of theseconventionally-raised mice fed the Western diet was dominated by thesame Mollicute lineage that had been identified in the earlierconventionalization experiment involving germ-free animals (FIG. 15).

The immune system is one of the host factors that influences gutmicrobial ecology [14-17]. However, this bloom occurred in all mice fedthe Western diet and did not require a functional innate or adaptiveimmune system: i.e., the Mollicute bloom was present at a significantlyhigher abundance in the cecal microbiota of conventionally-raisedWestern diet-fed C57BL/6J mice that were wild-type, MyD88−/− or Rag1−/−,compared to their genotypically-matched CHO-fed siblings (FIG. 15).

To directly test whether the DIO-associated gut microbial communitypossesses functional attributes that can increase host adiposity to agreater degree than a CHO-diet associated gut microbial community, wetransplanted the cecal microbiota harvested from obese,conventionally-raised wild-type donors who had been on the Western dietfor 8 weeks since weaning, or the cecal microbiota from lean CHO-fedcontrols, to 8-9 week-old germ-free CHO-fed recipients (n=1 donor and4-5 recipients/treatment group/experiment; n=3 independent experiments,including one CHO-fed control group described in ref. 2). All recipientswere maintained on a CHO diet (16% of kcal from fat, 61% fromcarbohydrates of which 2% are from fructose, glucose, lactose, maltose,and sucrose combined), and sacrificed 14d after receiving the microbiotatransplant (FIG. 11D). Mice colonized with a DIO-associated microbiotaexhibited a significantly greater percentage increase in body fat, asdefined by dual energy x-ray absorptiometry (DEXA), than mice who hadbeen gavaged with a microbiota from CHO-fed donors (43.0±7.1 versus24.8±4.9 percentage increase; p<0.05, Student's t-test based on thecombined data from all three experiments) (FIG. 13D). Importantly, therewere no statistically significant differences in chow consumption(14.5±0.3 versus 14.7±0.8 kcal/d) or initial weight (22.9±0.3 versus23.8±0.7 g) between recipients of the obese and lean cecal microbiotas.

To test the impact of defined shifts in diet on the body weight,adiposity, and distal gut microbial ecology of obese mice, we designedtwo custom chows that were modifications of the Western diet: one withreduced carbohydrates (CARB-R); the other with reduced fat (FAT-R) (seeTables 11 and 12 for information about the composition and caloricdensity of these diets). Sixteen conventionally-raised C57BL/6J mice,representing two families derived from two mothers who were sisters toensure that they all inherited a similar microbial community [8], wereweaned onto the Western diet and maintained on it for two months. Asubset of mice from each family was subsequently continued on theWestern diet for four weeks (n=5; control group), while the remainingsiblings were switched to the CARB-R (n=6), or FAT-R diets (n=5) forfour weeks (FIG. 11C).

Mice switched to the FAT-R or CARB-R diet consumed significantly fewercalories [12.5±0.1 kcal/d (FAT-R) and 12.0±0.2 kcal/d (CARB-R) versus14.1±0.2 kcal/d (Western); p<0.0001, ANOVA], gained significantly lessweight [0.6±0.3 g (FAT-R) and 0.0±0.3 g (CARB-R) versus 2.0±0.3 g(Western); p<0.01, ANOVA], and had significantly less fat [epididymalfat pad weight: 1.9±0.3% of total body weight for FAT-R and 1.9±0.2% forCARB-R versus 2.8±0.2% (Western); p<0.05, ANOVA] than those maintainedon the Western diet (FIG. 16). This provided us with the animal model wehad sought: diet-induced obesity followed by weight stabilization andreductions in adiposity, in genetically identical mice consuming defineddiets who had inherited a similar microbiota from their mothers.

16S rRNA gene sequence-based surveys revealed that weight stabilizationwas accompanied by (i) a significant reduction in the relative abundanceof the Mollicutes [31.9±11.6% of all bacterial sequences for FAT-R, anda significantly more pronounced decrease to 6.1±3.6% for CARB-R versus50.3±6.1% for the Western diet; p<0.05, ANOVA], and (ii) a significantdivision-wide increase in the relative abundance of Bacteroidetes(2.8-fold on the FAT-R, and 2.2-fold on the CARB-R diets; p<0.05, ANOVA)(FIG. 17).

To test if these alterations in gut microbial ecology had an effect onthe ability of the microbiota to promote host adiposity, we colonizedgerm-free, CHO-fed recipients with a cecal microbiota harvested fromconventionally-raised donors who had been on the Western diet sinceweaning (8 weeks) and then switched to a FAT-R or CARB-R diet (n=1 donorand 4-5 germ-free recipients/treatment group/experiment; n=2 independentexperiments;

FIG. 11D). Unlike with recipients of the DIO-associated microbiota,there was no statistically significant difference in the amount of fatgained between mice colonized with the FAT-R or CARB-R communities,compared to mice colonized with a cecal microbiota from lean CHO-feddonors (33.6±8.7%, 37.4±10.6%, and 24.8±4.9% increases, respectively;p=0.2, ANOVA).

Combined, these results indicate that both the FAT-R and CARB-R dietsrepress multiple effects of Western diet-induced obesity: i.e. theydecrease adipose tissue mass, diminish the bloom in a single unculturedMollicute lineage, increase the relative abundance of Bacteroidetes, andreduce the ability of the microbiota to promote fat deposition.

Example 7 Western Diet-Associated Gut Microbiome

To further investigate the linkage between diet-induced obesity and theMollicute bloom, we performed capillary sequencing of seven cecalsamples obtained from seven mice: (i) three samples were from animalsfed the Western diet (one that had been conventionalized, two that wereconventionally-raised), (ii) two were from conventionally-raised micethat had been switched from the Western to FAT-R diet for 4 weeks, and(iii) two were from conventionally-raised mice that had been switched tothe CARB-R diet for 4 weeks (one mouse/family/diet; as noted above, theconventionally-raised mice were from two mothers who were sisters; Table14). A total of 48 Mb of high-quality sequence data was generated(average of 7 Mb/cecal DNA sample; Table 15).

TABLE 14 Nomenclature used to designate microbiome datasets obtainedfrom the cecal microbiota of C57BL/6J mice Host 16S rRNA Figure labelMicrobiome label family Host state Host diet survey label ARB labelWestern 1 WEST1 1 CONV-R Western Western 10 MD22 FAT-R 1 FATR1 1 CONV-RFAT-R FAT-R 4 MD25 CARB-R 1 CARBR1 1 CONV-R CARB-R CARB-R 2 MD8 Western2 WEST2 2 CONV-R Western Western 9 MD20 FAT-R 2 FATR2 2 CONV-R FAT-RFAT-R 5 MD27 CARB-R 2 CARBR2 2 CONV-R CARB-R CARB-R 4 MD21 Western 3WEST3 — CONV-D Western Western 3 WD8

TABLE 15 Microbiome sequencing statistics Microbiome Average read lengthForward reads^(a) Sequence (Mb) Western 1 668 9,072 6.1 FAT-R 1 58610,681 6.3 CARB-R 1 603 10,773 6.5 Western 2 633 10,997 7.0 FAT-R 2 72310,893 7.9 CARB-R 2 591 10,244 6.1 Western 3 734 11,705 8.6 TOTAL —74,365 48 ^(a)trimmed according to quality and vector sequence

Taxonomic Assignments

All seven datasets were dominated by sequences homologous to knownbacterial genomes (49.97±2.52%), followed by sequences with nosignificant homology to any entries in the non-redundant (NR) database(34.82±1.89%) or that could not be confidently assigned (10.28±0.45%),followed by sequences homologous to eukarya (4.56±1.02%), archaea(0.27±0.05%), and viruses (0.10±0.01%) (BLASTX assignments performedwith MEGAN [18]; for further details see methods; FIG. 18A). Thesequences homologous to eukarya could be assigned to two principalgroups: metazoa (largely derived from host cells) and apicomplexa.

Consistent with the PCR-based 16S rRNA data, the largest group ofsequences in all seven cecal microbiomes was homologous to theFirmicutes division of Bacteria. Analysis of 16S rRNA gene fragmentsculled from the metagenomic datasets confirmed the presence of theMollicute bloom in the Western diet-associated cecal microbiome (FIG.18D). However, all of the datasets, including those from mice on theWestern diet, had a low relative abundance of sequences homologous topreviously sequenced Mollicute genomes (FIG. 18C). These results supportthe conclusion that the genetic make-up of the DIO-associated Mollicutebloom is distinct from that of previously sequenced Mollicutes.

Analysis of 16S rRNA gene fragments and NR-based taxonomic assignmentsconfirmed that both the FAT-R and the CARB-R diets resulted in anincreased relative abundance of sequences homologous to theBacteroidetes (FIG. 18B,D). To focus on the microbiomes' bacterial andarchaeal gene content, all sequences that could be confidently assignedto eukarya were removed before conducting the analyses described below.

Functional Predictions

Metagenomic sequencing reads were subsequently assigned to orthologousgroups from the STRING-extended COG database [19] and the KyotoEncyclopedia for Genes and Genomes (KEGG) [20]. KEGG pathway-basedmetabolic reconstructions of cecal microbiomes harvested from mice fedthe Western, CARB-R, or FAT-R diets revealed a variety of differencesassociated with the various diets (Table 16). Notably, the Western dietmicrobiome is significantly enriched for KEGG pathways involved in theimport and fermentation of simple sugars and host glycans, including‘fructose and mannose metabolism’ and ‘phosphotransferase system’(p<0.05 based on bootstrap analysis of pathways in the Westerndiet-versus CARB-R microbiomes).

TABLE 16 KEGG pathways significantly enriched or depleted in the Westerndiet microbiome* KEGG pathway Enriched Phosphotransferase system (PTS)Fructose and mannose metabolism Glycolysis/Gluconeogenesis Glutamatemetabolism Carbon fixation Unclassified (non-enzyme) Pyrimidinemetabolism Protein export Phenylalanine, tyrosine and tryptophanbiosynthesis Oxidative phosphorylation Depleted ABC transportersBacterial chemotaxis Bacterial motility proteins Flagellar assemblyProtein kinases Two-component system Pentose and glucuronateinterconversions Other amino acid metabolism Starch and sucrosemetabolism Ribosome *Based on bootstrap analysis of pathway relativeabundance in the Western versus CARB-R microbiome (p < 0.05)

Phosphotransferase systems (PTS) are a class of transport systemsinvolved in the uptake and phosphorylation of a variety of carbohydrates[21]. Each transporter involves three linked enzymes that act asphosphoryl group recipients and donors: two are cytoplasmic enzymes thatact on all imported PTS carbohydrates (HPr and EI); the other is acarbohydrate-specific complex (EII) comprising one or two hydrophobicintegral membrane domains (EIIC/D) and two hydrophilic domains (EIIA/B)[21]. Phosphoenolpyruvate, produced though glycolysis, can be used togenerate ATP (via pyruvate kinase), or used to drive the import ofadditional sugars through transfer of a phosphoryl group to EI of thePTS (FIG. 19). PTS genes are found in multiple divisions of bacteria,including Proteobacteria such as E. coli, as well as multiple sequencedFirmicutes (e.g., the Mollicutes Mycoplasma genitalium, M. pneumoniae,M. pulmonis, M. penetrans, M. gallisepticum, M. mycoides, M. mobile, M.hyopneumoniae, M. synoviae, and M. capricolum; KEGG version 40) [20].The PTS also plays a role in regulating microbial gene expressionthrough catabolite repression, allowing the cell to preferentiallyimport simple sugars over other carbohydrates [21].

Multiple components of the PTS are present in the Western dietmicrobiome (EI and HPr plus EII), which could allow the import of simplesugars (e.g., glucose and fructose that together comprise sucrose, anabundant component of the Western diet), as well as sugars associatedwith the host gut mucosa (N-acetyl-galactosamine) (FIG. 19). The Westerndiet microbiome also contains genes that support metabolism of thesephosphorylated sugars to various end-products of anaerobic fermentation(e.g. lactate and the short-chain fatty acids butyrate and acetate; FIG.4). In addition, the Western diet microbiome is enriched for genesencoding beta-fructosidase, a glycoside hydrolase capable of fermentingbeta-fructosidases such as sucrose, inulin, or levan (p<0.05 based on aχ² test of Western versus CARB-R microbiome).

The Western diet-associated cecal microbiome contains genes for cellwall biosynthesis and cell division: (i) orthologous groups COG0707,COG0766, and COG0768-COG0773 (together, found at a slightly higherrelative abundance in the Western versus CARB-R microbiome; p=0.3 basedon a χ² test); (ii) multiple components of the KEGG pathway forpeptidoglycan biosynthesis; and (iii) all enzymes in the2-methyl-D-erythritol 4-phosphate (MEP) pathway that converts pyruvateto isopentyl-pyrophosphate (IPP; FIG. 19). IPP provides, among otherthings, a precursor for peptidoglycan biosynthesis [with the aid ofgenes for farnesyl diphosphate synthase (KO0795) and undecaprenyldiphosphate synthetase (KO0806) that were also identified in themicrobiome]. Together, these findings indicate that unlike otherMollicutes (e.g., the mycoplasmas), members of the bloom have thecapacity to construct a cell wall.

Additionally, unlike the more diverse Firmicutes-enriched ob/ob andCARB-R microbiomes, the Western diet-associated microbiome is depletedfor genes assigned to KEGG pathways involved in motility, including (i)‘bacterial chemotaxis’, (ii) ‘bacterial motility proteins’, and (iii)‘flagellar assembly’ (Table 16). This observation suggests that theMollicute bloom is either non-motile or utilizes a mechanism for glidingmotility, such as that found recently in other Mollicutes, that isindependent of the known pathways for bacterial chemotaxis and flagellarbiosynthesis [22-23].

Assembly and Analysis of Contigs

All seven microbiome datasets were assembled individually and as onepooled dataset using the program ARACHNE [24]. As expected, the reduceddiversity of the Western diet microbiome produced the largest contiguous‘genome fragments’ (Table 17). Manual inspection of genome fragmentsfrom the combined assembly (N50 contig length=1738 bases; FIG. 20),revealed multiple contigs containing genes that were enriched in theWestern diet microbiome, including those involved in the degradation ofbeta-fructosides such as sucrose, inulin, and levan (fructanbeta-fructosidase) and the import of simple sugars (PTS genes forfructose and glucose transport). A large contig was also found thatcontained multiple genes involved in the import of amino acids (ABCtransporters) (FIG. 20). Interestingly, the two genome fragmentscontaining PTS genes were each flanked by another gene involved incarbohydrate metabolism: in one case, an alpha-amylase (starchdegradation) and in the other fragment, fructose-bisphosphate aldolase(glycolysis). These genome fragments are likely derived from theexpanded uncultured Mollicute Glade: they are composed of reads frommicrobiomes with a high relative abundance of the bloom and share thehighest degree of homology with Bacillus and Mollicute genomes (Table18).

TABLE 17 Microbiome assembly statistics Mean trimmed Fraction Number N50Max Input read assembled of contig contig Sample reads length (%)contigs length length Western 1 11136 612 0.9 17 1306 2159 FAT-R 1 12288582 0.2 6 1218 1458 CARB-R 1 12288 590 0.0 2 1246 1246 Western 2 11904573 2.6 37 1782 7376 FAT-R 2 11904 622 0.8 23 1428 3451 CARB-R 2 11520575 0.1 4 1071 1236 Western 3 13440 627 6.7 107 1884 11022 Allmicrobiomes 84480 598 3.9 387 1738 11990

TABLE 18 Read placements in contigs and BLAST results Western WesternWestern FAT-R FAT-R CARB-R CARB-R 1 2 3 1 2 1 2 BBH^(a) e-value contig235 5 9 4 1 0 0 S. mutans 0 contig73 1 0 2 0 0 0 0 S. mutans 4E−91 contig146 2 4 4 3 1 0 0 E. faecalis 0 contig161 2 1 4 3 3 0 0 E.dolichum 3E−97  contig262 1 5 0 3 4 0 0 L. monocytogenes 1E−119

Validation of PTS Expression

We constructed a cDNA library from mRNA enriched total community RNAthat had been isolated from the cecum of an obese mouse fed the Westerndiet (see Materials and Methods from example 7 for details regarding themRNA enrichment procedure). Sequence analysis of the inserts in thislibrary confirmed that a gene encoding E11 of the fructose, mannose, andN-acetylgalactosamine specific PTS transporter (COG3716) was expressed.The low representation of mRNA-derived sequences in our libraryprecluded further (costeffective) characterization of the DIO cecalmicrobiome's transcriptome. However, sequencing of 16S rRNA-derivedinserts in the library provided further support of the high abundance ofthe Mollicute bloom: 80.6% of expressed 16S rRNAs had a best-BLAST-hitto Mollicute gene sequences (BLASTN comparisons with the NCBI nucleotidedatabase, e-value <10⁻²⁵).

Biochemical Validation of Enhanced Fermentation in the DIO Microbiota

To verify our in silico predictions concerning metabolic activities thatare enriched in the Western-diet associated gut microbiome, we performedgas-chromatography-mass spectrometric and microanalytic biochemicalassays of the concentrations of short chain fatty acids and lactate inaliquots of the same cecal samples that had been used for 16S rRNAsurveys and metagenomic sequencing of community DNA (See Methods).

Biochemical Analysis

Short-chain fatty acids (SCFAs) were measured in cecal samples obtainedfrom mice fed Western, FAT-R, or CARB-R diets (n=3-5 mice/group; twoaliquots per mouse). The procedure, described in an earlier publication[49], involved double diethyl ether extraction of deproteinized cecalcontents spiked with isotope-labeled internal SCFA standards (Isotec:[²H₃]- and [2⁻¹³C]acetate, [²H₅]propionate, and [¹³C₄]butyrate),derivatization of SCFAs withN-tert-butyldimethylsilyl-N-methyltrifluoracetamide (MTSTFA), and GC-MSanalysis of the resulting TBDMS-derivatives using a gas chromatograph(Model 6890; Hewlett-Packard) interfaced to a mass spectrometer detector(Model 5973; Agilent Technologies).

Lactate levels were quantified using a microanalytic approach: cecalsamples were quick frozen in liquid nitrogen, stored at −80° C., andlyophilized at −35° C. 1-5 mg of dried cecal material was homogenized in0.4 ml 0.2 M NaOH at 1° C. Alkali extracts were prepared by heating an80 μl aliquot for 20 min at 80° C. and adding 80 μl of 0.25 M HCl and100 mM Tris base. Acid extracts were prepared by adding 20 μl 0.7 M HClto a separate 60 μl aliquot, heating for 20 min at 80° C., andneutralizing with 40 μl of 100 mM Tris base. The Bradford method wasused to determine the protein content of the alkali extracts (BioRad).Cecal lactate levels were determined using a combination of pyridinenucleotide coupled enzymatic reactions with the Lowry oil well techniqueand enzymatic cycling amplification [50]. A 0.2 μl aliquot (25-100 ngprotein) from the acid extracts was added to 2 μl of reagent containing50 mM 2-Amino-2-methanol-1-proponal buffer pH 9.9, 2 mM glutamate pH9.9, 0.2 mM NAD+, 50 ug/ml beef heart lactate dehydrogenase (Sigma;specific activity 500 units/mg protein) and 50 μg/ml pig heart glutamatepyruvate transaminase (Roche; spec. act. 80 units/mg protein). Followinga 30 min incubation at 24° C. the reaction was terminated with theaddition of 1 μl 0.15M NaOH and heated 20 min at 80° C. Once the samplescooled to 24° C., a 1 μl aliquot was transferred to 0.1 ml NAD cyclingreagent and amplified 5000 fold. Lactate standards, 5 to 10 μM, werecarried throughout all steps.

As predicted from our metabolic reconstructions, the cecal contents ofmice fed the Western diet (on average, 50% Mollicutes) had asignificantly higher concentration of multiple end-products of bacterialfermentation, including lactate, acetate, and butyrate compared to thececal contents of CARB-R mice (on average, 6% Mollicutes) (FIG. 21).

Example 8 Whole Genome Sequencing and Analysis of a Human Gut-AssociatedMollicute

Representatives of the Mollicute Glade that blooms in the distal gutmicrobiota of mice fed a Western diet have yet to be successfullycultured. Therefore, to obtain additional insights about genomic andmetabolic features that may allow this lineage to bloom in the cecalhabitat of mice fed a Western diet, and to validate our comparativemetagenomic predictions, the genome of Eubacterium dolichum strainATCC29143, a related Mollicute (FIG. 14) isolated from the human gutmicrobiota (Table S8) was sequenced.

Whole Genome Sequencing and Annotation

A draft assembly of the Eubacterium dolichum strain ATCC29143 genome wasgenerated from ABI 3730x1 paired-end reads of inserts in whole genomeshotgun plasmid libraries (35,683 reads; average read length of 569nucleotides, representing—9× coverage), as well as from reads producedfrom one run of the 454 FLX pyrosequencer (425,423 reads with meanlength of 250 nucleotides, representing—49× coverage).

The Newbler de novo shotgun sequence assembler was used to assemble 454FLX sequences based on flowgram signal space. This process includesoverlap generation, contig layout, and consensus generation. Theresulting contigs were then broken into linked sequences to generatepseudo paired-end reads, and aligned with 3730x1 reads using PCAP [45].To minimize potential assembly/contamination errors in the draftgenomes, only contigs greater than 2 kb were used. Genes were predictedusing MetaGene [25]. Each predicted gene sequence was translated, andthe resulting protein sequence assigned InterPro numbers usingInterProScan (version 4.3) [31]. Each gene was annotated based on theoutput of InterProScan and BLASTP comparisons versus the KEGG database(version 40) [20] and the STRING database (version 7) [19], in additionto experimentally validated metabolic pathway maps in the MetaCycdatabase (metacyc.org) [46].

For KEGG pathway analysis, the relative abundance each pathway wascalculated for each genome (number of genes assigned to a given pathwaydivided by the total number of pathway assignments). The relativeabundance was then converted into a z-score based on the mean andstandard deviation of the given pathway across all microbiomes. KEGGpathways were clustered using Cluster3.0 [47]. Single linkagehiearchical clustering via Euclidean distance was performed, and theresults visualized (Treeview Java applet) [48].

A deep draft assembly of its genome was produced, based on 49-foldcoverage with reads from a 454 FLX pyrosequencer (106 Mb), and 9-foldcoverage with reads from a traditional ABI 3730x1 capillary sequencer(GenBank accession ABAWO0000000; genome.wustl.edu/pub/organism/).

TABLE 19 E. dolichum draft genome sequencing statistics Total contignumber 51 Total contig bases 2209242 Average contig length 43318 Maximumcontig length 453733 N50 contig length 291535 N50 contig number 3 Majorcontig (>2000 bp) number 17 Major contig bases 2181491 GC content 38

We first compared this deep draft assembly of the E. dolichum genome toeight other deep-draft assemblies of human gut-associated Firmicutes andto fourteen finished Mollicute genomes (FIGS. 22 and 23). The programMetaGene [25] was used to predict the protein products of these diverseFirmicute/Mollicute genomes and the proteins assigned to theSTRING-extended COG database [19] and the KEGG database [20] usingBLASTP homology searches (e-value <10⁻⁵).

Principal component analysis (PCA) of KEGG pathway representation in all23 genomes revealed a clear clustering of the previously sequencedMollicute genomes and the recently sequenced commensal gut Firmicutes,including E. dolichum (FIG. 22A). The total size of the E. dolichumassembly is over twice the average Mollicute genome (2.2 versus 0.91Mb), and two-thirds the average size of the recently sequenced gutFirmicute genomes (3.2 Mb). Our analyses revealed that the genome sizereduction and corresponding gene loss that has occurred during Mollicuteevolution has produced small genomes that are largely restricted toencoding components of metabolic pathways essential for life (FIG. 24).Accordingly, bacterial genome size significantly correlates with theclustering results (FIG. 18B; R²=0.9, p<0.05). As expected from itsrelatively restricted genome size, E. dolichum is enriched for many KEGGpathways involved in essential cellular functions such as “Celldivision”, “Replication, Recombination, and Repair”, “Ribosome”, andothers (FIG. 23) but is missing a number of metabolic pathways similarto other ‘streamlined’ genomes (e.g. the mycoplasma, and oceanicα-proteobacteria) [22,26]. Its genome lacks predicted proteins involvedin bacterial chemotaxis and flagellar biosynthesis, the tricarboxylicacid cycle, the pentose phosphate cycle, and fatty acid biosynthesis(FIG. 22C). It is also significantly depleted for ABC transportersrelative to the other gut Firmicutes (FIG. 23), and a variety ofmetabolic pathways for the de novo synthesis of vitamins and amino acidsare incomplete or undetectable (FIG. 22C).

E. dolichum has a number of genomic features that could promote fitnessin the cecal nutrient metabolic milieu created by the host's consumptionof the Western diet. As in the metagenomic dataset generated from theWestern diet-associated cecal microbiome, its genome is enriched forpredicted PTS proteins involved in the import of simple sugars includingglucose, fructose, and N-acetyl-galactosamine (FIGS. 19 and 23).STRING-based protein networks constructed from the E. dolichum genomerevealed that many of these PTS orthologous groups are found in theWestern diet microbiome, but not in all nine recently sequenced gutFirmicutes (FIG. 24). In addition, the E. dolichum genome encodes abeta-fructosidase capable of degrading fructose-containing carbohydratessuch as sucrose, genes for the metabolism of PTS-imported sugars tolactate, butyrate, and acetate, plus a complete 2-methyl-D-erythritol4-phosphate pathway for isoprenoid biosynthesis—all genetic features ofthe Western-diet-associated cecal microbiome (FIGS. 19 and 24).

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What is claimed is:
 1. A method for modulating body fat, for modulatingbody weight, or for modulating energy harvest comprising: altering themicrobiota population in the gastrointestinal tract of a subject in needof body fat, body weight, or energy harvest modulation by altering thediversity of the microbiota population, altering the relative abundanceof a Bacteroidetes species, altering the relative abundance of aFirmicutes species, or altering the ratio of a Firmicutes species to aBacteroidetes species, whereby alteration of the diversity of themicrobiota population, or the relative abundance or ratio of saidBacteroidetes or Firmicutes species modulates body fat, body weight orenergy harvest in said subject.
 2. The method of claim 1, wherein themethod comprises administering a particular diet or diet ingredient, aprobiotic, an antibiotic or a therapeutic compound to the subject. 3.The method of claim 1, wherein the method comprises administering tosaid subject a probiotic comprising a Firmicutes or a Bacteroidetes, ora combination thereof.
 4. The method of claim 3, wherein the probioticcomprises a species listed in Table A.
 5. The method of claim 1, whereinthe method comprises administering an antibiotic that is moreefficacious against either Firmicutes or Bacteroidetes as compared tothe other.
 6. The method of claim 1, wherein the method comprisesadministering a particular diet or diet ingredient that supports theestablishment and functions of either Firmicutes or Bacteroidetes in themicrobiota.
 7. The method of claim 2, wherein the particular diet is ahigh-fat/high-sugar (“Western”) diet, a restricted calorie diet, areduced carbohydrate diet, a reduced fat diet, a reduced protein diet,or a low-fat high-polysaccharide chow diet.
 8. The method of claim 1,comprising increasing the subject's body fat, body weight or energyharvest, wherein the diversity of the microbiota population isdecreased, the relative abundance of Bacteroidetes is decreased, therelative abundance of Firmicutes is increased or the ratio of Firmicutesto Bacteroidetes is increased.
 9. The method of claim 8, wherein therelative abundance of Bacteroidetes is decreased by about 1% to about100% or the relative abundance of Firmicutes is increased by about 1% toabout 100%.
 10. The method of claim 8, wherein the method comprisesadministering a probiotic comprising a Firmicutes species to thesubject.
 11. The method of claim 8, wherein the method comprisesadministering an antibiotic to the subject, wherein the antibiotic ismore efficacious against Bacteroidetes as compared to Firmicutes. 12.The method of claim 8, wherein the method comprises administering ahigh-fat/high-sugar (“Western”) diet to the subject.
 13. The method ofclaim 1, comprising decreasing the subject's body fat, body weight orenergy harvest, wherein the biodiversity of the microbiota population isincreased, the relative abundance of Bacteroidetes is increased, therelative abundance of Firmicutes is decreased or the ratio of Firmicutesto Bacteroidetes is decreased.
 14. The method of claim 13, wherein therelative abundance of Bacteroidetes is increased by about 1% to about100% or the relative abundance of Firmicutes is decreased by about 1% toabout 100%.
 15. The method of claim 13, wherein the method comprisesadministering a probiotic comprising a Bacteroidetes species to thesubject.
 16. The method of claim 13, wherein the method comprisesadministering an antibiotic to the subject, wherein the antibiotic ismore efficacious against Firmicutes as compared to Bacteroidetes. 17.The method of claim 13, wherein the method comprises administering arestricted calorie diet, a reduced carbohydrate diet, a reduced fatdiet, a reduced protein diet, or a low-fat high-polysaccharide chowdiet.
 18. The method of claim 1, wherein the subject is a rodent, ahuman, a livestock animal, a companion animal, or a zoological animal.19. The method of claim 1, wherein the subject is a livestock animal.20. The method of claim 19, wherein the subject is a pig, cow, horse,goat, or sheep.
 21. A method of claim 1, wherein the method comprisesdetermining if a subject is in need of body fat, body weight, or energyharvest modulation.
 22. A method for modulating body fat, for modulatingbody weight, or for modulating energy harvest comprising: (a)determining if a subject is in need of body fat, body weight, or energyharvest modulation; and (b) altering the diversity of the microbiotapopulation, altering the relative abundance of a Bacteroidetes species,altering the relative abundance of a Firmicutes species, or altering theratio of a Firmicutes species to a Bacteroidetes species, wherebyalteration of the diversity of the microbiota population, or therelative abundance or ratio of said Bacteroidetes or Firmicutes speciesmodulates body fat, body weight or energy harvest in said subject.